High Throughput Quantitative Microscopy System

ABSTRACT

Methods and systems are provided for an imaging system. In one example, the imaging system includes a plurality of microscope assemblies arranged radially around a central axis of the imaging system, coupled to vertically oriented plates with a set of objectives arranged at tops of the plurality of microscope assemblies and wherein the plates are oriented to extend away from the central axis in a radial direction to form an x-shaped configuration.

FIELD

The present description relates generally to methods and systems forhigh throughput quantitative microscopy.

BACKGROUND/SUMMARY

Application of microscopy to high throughput screening has become apowerful and efficient method for drug development, as well as otherbiological and chemical experimentation. As such, microscopy may be usedto extract quantitative data from images, in contrast to earlyapplications when microscopy only allowed for qualitative analysis. Inparticular, when quantitative microscopy is paired with sample screeningusing microplates (e.g., microtiter plates), multiple samples may beanalyzed concurrently, thereby expediting sampling throughput.

With current efforts to develop cutting-edge treatments to existing andnewly-discovered conditions, medical, biological, and chemical advancesmay be hindered by a rate at which samples can be screened. More rapidprocessing and analysis of samples enables examination of more compoundsand/or conditions, expediting discovery of new results and findings.Furthermore, faster screening may allow time-dependent biologicalprocesses to be observed, thereby providing more useful and relevantinformation. In particular, application of accelerated screening tostudying live cells may enable generation of high quality, accuratemodels for drug discovery.

For example, a microscopic system configured to provide high throughputdata may process samples at a speed dependent on a field-of-view of themicroscope objective at a desired magnification and resolution. Eachimage collected by the system may capture only a small region of a wellof a microplate, thereby demanding numerous imaging cycles beforescreening of the microplate is complete. However, observation of rarebiological events and findings, upon which advances in drug treatmentmay depend, may require processing of large numbers of cells. Forexample, identification of a single cell with a distinct phenotype mayonly occur upon screening at least one million cells. As such, morevaluable information may be obtained from faster screening of onemicroplate-supported sample rather than high throughput screening ofmany different samples.

In one example, the issues described above may be addressed by animaging assembly, comprising a plurality of microscope assembliesarranged radially around a central axis of the imaging system, coupledto vertically oriented plates with a set of objectives arranged at topsof the plurality of microscope assemblies and wherein the plates areoriented to extend away from the central axis in a radial direction toform an x-shaped configuration. In this way, indexing of a microplatemay be executed at a high frequency, thereby enabling capture oftransient signal pathways for live specimens.

It should be understood that the summary above is provided to introducein simplified form a selection of concepts that are further described inthe detailed description. It is not meant to identify key or essentialfeatures of the claimed subject matter, the scope of which is defineduniquely by the claims that follow the detailed description.Furthermore, the claimed subject matter is not limited toimplementations that solve any disadvantages noted above or in any partof this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of a quantitative microscopy assembly.

FIG. 2 shows a first perspective view of a multi-detector quantitativemicroscopy system.

FIG. 3 shows a front view of a blade of the multi-detector quantitativemicroscopy system of FIG. 2 .

FIG. 4 shows a front view of inner components of the blade of FIG. 3 .

FIG. 5 shows a rear view of the blade.

FIG. 6 shows an example of a microplate which may be used in themulti-detector quantitative microscopy system.

FIG. 7 shows a top view of a plate holder which may be used to securethe microplate of FIG. 6 to the multi-detector quantitative microscopysystem.

FIG. 8 shows a top view of the plate holder with the microplate removed.

FIG. 9 shows a portion of the plate holder in greater detail.

FIG. 10 shows a bottom view of the plate holder.

FIG. 11A shows a view of the plate holder with a mechanism for movingthe plate holder in a first position.

FIG. 11B. shows a view of the plate holder with the mechanism for movingthe plate holder in a second position.

FIG. 12 shows a top view of the multi-detector quantitative microscopysystem with the plate holder removed to show objectives of themulti-detector quantitative microscopy system.

FIG. 13 shows a front view of the multi-detector quantitative microscopysystem.

FIG. 14 shows a second perspective view of the multi-detectorquantitative microscopy system.

FIG. 15 shows a lower portion of the multi-detector quantitativemicroscopy system.

FIG. 16 shows a top view of a central fan of the multi-detectorquantitative microscopy system.

FIG. 17 shows an example of a field of view of an objective of themulti-detector quantitative microscopy system overlaid with a grid.

FIG. 18 shows a schematic diagram of the objective position under a wellof the microplate.

FIG. 19 shows an example of a conventional light shape used in anautofocus system for a microscope.

FIG. 20 shows an example of a light shape used in an autofocus system ofthe multi-detector quantitative microscopy system.

FIG. 21 shows a first plot showing a focusing of a microscope using thelight shape of FIG. 19 .

FIG. 22 shows a second plot showing a focusing of an objective of themulti-detector quantitative microscopy system using the light shape ofFIG. 20 .

FIG. 23 shows a diagram illustrating a positioning of an objectiveduring an autofocusing method of the multi-detector quantitativemicroscopy system.

FIG. 24 shows a flow diagram of the autofocusing method of FIG. 23 .

FIGS. 25A-25B show an example of a method for collecting images of themicroplate by via the multi-detector quantitative microscopy system.

FIG. 26 shows an example of a method for coordination illumination ofthe microplate by light sources of the multi-detector quantitativemicroscopy system, which may be executed in conjunction with the methodof FIGS. 25A-25B.

FIGS. 2-16 are shown approximately to scale.

DETAILED DESCRIPTION

The following description relates to systems and methods for highthroughput quantitative microscopy. Quantitative microscopy may be usedto extract information from digital images by illuminating a sample witha desired wavelength, or range of wavelengths, of light. In one example,the wavelength may be selected to induce fluorescence from the samplewhich may be measured by a detector to provide a quantitative analysisof sample properties. An example of an assembly for quantitativemicroscopy is depicted in FIG. 1 as a schematic diagram. The assemblyshown in FIG. 1 may be included in each of four blades of amulti-detector quantitative microscopy system, as shown in FIG. 2 . Eachof the blades includes an individual quantitative microscopy assemblyand various views of one of the blades is shown in FIGS. 3-5 ,illustrating both external and internal components. The multi-detectorquantitative microscopy system may be configured to collect images ofsamples which may be supported in a microplate, as depicted in FIG. 6 .The microplate may be coupled to the multi-detector quantitativemicroscopy system by a plate holder, as shown in FIGS. 7-11B. Apositioning of objectives at a top of the multi-detector quantitativemicroscopy system is shown in FIG. 12 and different views of themulti-detector quantitative microscopy system are illustrated in FIGS.13-14 . A cooling system of the multi-detector quantitative microscopysystem may include a central fan as illustrated in FIGS. 15-16 . Themulti-detector quantitative microscopy system may utilize an autofocussystem to focus the objectives relative to the microplate. Details ofthe autofocus system are shown, including a grid overlaid with a fieldof view of an objective at FIG. 17 , a positioning of the objectiverelative to the microplate at FIG. 18 , light shapes used forautofocusing at FIGS. 19-20 , plots showing the focusing of theobjective at FIGS. 21-22 , and a method for the autofocusing at FIGS.23-24 . An example of a method for operating the multi-detectorquantitative microscopy system to obtain images of the microplate isshown in FIGS. 25A-25B which further includes a method for synchronizinglight sources of the multi-detector quantitative microscopy system toilluminate the microplate during image collection.

FIGS. 2-16 show example configurations with relative positioning of thevarious components. If shown directly contacting each other, or directlycoupled, then such elements may be referred to as directly contacting ordirectly coupled, respectively, at least in one example. Similarly,elements shown contiguous or adjacent to one another may be contiguousor adjacent to each other, respectively, at least in one example. As anexample, components laying in face-sharing contact with each other maybe referred to as in face-sharing contact. As another example, elementspositioned apart from each other with only a space there-between and noother components may be referred to as such, in at least one example. Asyet another example, elements shown above/below one another, at oppositesides to one another, or to the left/right of one another may bereferred to as such, relative to one another. Further, as shown in thefigures, a topmost element or point of element may be referred to as a“top” of the component and a bottommost element or point of the elementmay be referred to as a “bottom” of the component, in at least oneexample. As used herein, top/bottom, upper/lower, above/below, may berelative to a vertical axis of the figures and used to describepositioning of elements of the figures relative to one another. As such,elements shown above other elements are positioned vertically above theother elements, in one example. As yet another example, shapes of theelements depicted within the figures may be referred to as having thoseshapes (e.g., such as being circular, straight, planar, curved, rounded,chamfered, angled, or the like). Further, elements shown intersectingone another may be referred to as intersecting elements or intersectingone another, in at least one example. Further still, an element shownwithin another element or shown outside of another element may bereferred as such, in one example.

Turning now to FIG. 1 , a schematic diagram for a quantitativemicroscopy assembly 100 (hereafter, the assembly 100) is shown. In oneexample, the assembly 100 may be configured as a fluorescence microscopyassembly 100. However, various other types of analytical microscopicimaging techniques are possible, including but not limited toluminescence, colorimetry, etc. The assembly 100 of FIG. 1 includes alight source 102 providing incident light to components arranged in apath of the incident light, as indicated by arrow 104. The light source102 may be a mercury-vapor lamp, a xenon arc lamp, a laser, or one ormore light-emitting diodes (LEDs).

The incident light may be directed to a filter cube (or filter block)106. The filter cube 106 may house components that filter the incidentlight such that target wavelengths are transmitted to a target to beanalyzed, e.g., one or more samples supported on a sample holder 108. Inone example, the sample holder 108 may be a microplate. Three filteringcomponents are arranged in the filter cube 106, including an excitationfilter 110, a dichroic filter 112, and an emission filter 114. Theincident light may first pass through the excitation filter 110 whichfilters the light to allow only select, e.g., target, wavelengths tocontinue past the excitation filter 110. The target wavelengths may bewavelengths that excite electrons in specific fluorophores orfluorochromes, resulting in release of photons when the excitedelectrons relax to a ground state. The excitation light, e.g., lightthat has been filtered by the excitation filter 110, then strikes thedichroic filter (or dichroic beamsplitter) 112, as indicated by arrow116. The dichroic filter 112 may be a mirror, for example, arranged at a45 degree angle relative to an optical path of the assembly 100, e.g.,angled at 45 degrees relative to the path of incident light indicated byarrow 104.

A surface of the dichroic filter 112 may include a coating that reflectsthe excitation light, e.g., light filtered by the excitation filter 110but allows fluorescence emitted from the sample at the sample holder 108to pass therethrough. The reflected excitation light, as indicated byarrow 116, passes through an objective 118 to illuminate the sampleholder 108. If the sample fluoresces, light is emitted, e.g., generatingemission light as indicated by arrow 120, and collected by the objective118. The emission light passes through the dichroic filter 112 andcontinues to the emission filter 114 which blocks undesired excitationwavelengths. The filtered emission light is received at a detector 122.The detector 122 may be a camera, such as a charge-coupled device (CCD)camera, in one example. In other examples, the detector 122 may beanother type of camera, for example, a CMOS camera, or a photomultipliertube.

At the detector 122, the emission light may be converted into electronicdata. For example, when the detector 122 is the CMOS camera, thedetector 122 may include a light sensor configured as a transistor on anintegrated circuit. Photons of the emission light may be incident on thelight sensor and generate an electrical charge that is converted intoelectronic data representative of a photon pattern of the emission lightcaptured within a field of view (FOV) of the camera. The electronic datamay be stored at a memory of the camera, such as random access memory,and may be retrieved by a controller 124.

The controller 124 may be a computer, including various components suchas a processor, input/output ports, an electronic storage medium forexecutable programs and calibration values, random access memory, a databus, etc. The electronic storage medium can be programmed with computerreadable data representing instructions executable by the processor forperforming the methods described below as well as other variants thatare anticipated but not specifically listed. The controller 124 may becoupled to various accessory devices including input devices such as akeyboard, a mouse, etc.

The controller 124 may be communicatively coupled to components of theassembly 100. For example, the controller 124 may be configured tocommand activation/deactivation of the light source 102 when promptedbased on user input. As another example, the controller 124 may instructadjustment of a position of the sample holder 108 to focus theexcitation light on a different region of the sample holder. Thecontroller 124 may command actuation of a motor 126 coupled to thesample holder 108 to vary the position of the sample holder 108 withrespect to the objective 118 and the excitation light and provideinstructions on how the sample holder position is to be modified. Insome examples, a position sensor 128 may monitor the actual position ofthe sample holder 108 and may be communicatively coupled to thecontroller 124 to relay the sample holder position to the controller124.

The controller 124 may also be communicatively coupled to the detector122. As such, electronic data collected by the detector 122 may beretrieved by the controller 124 for further processing and display at aninterface, such as a computer monitor. It will be appreciated that thecontroller 124 may be further coupled to other sensors and actuators ofthe assembly 100. In one example, communication between the controller124 and the sensors and actuators of the assembly 100 may be enabled byvarious electronic cables, e.g., hardwiring. In other examples, thecontroller 124 may communicate with the sensors and actuators via awireless protocol, such as Wifi, Bluetooth, Long Term Evolution (LTE),etc.

The assembly 100 may further include an auto-focus system 130,communicatively coupled to the controller 124. The auto-focus system 130may utilize a sensor configured with a light source and optical elementsfor modifying and directing a light beam from the light source to thesample holder 108. An image may be generated based on reflection of thelight beam which may be used by the controller 124 to determine asuitable adjustment of the objective and/or the sample holder 108 toalign the focus of the objective with a target interface at the sampleholder 108. In one example, the auto-focus system 130 may rely on alaser beam, as described below, with reference to FIGS. 17-23 , andauto-focus algorithms implemented at the controller 124 to rapidly focusthe assembly 100 on a desired area of the sample.

It will be appreciated that the assembly 100 depicted in FIG. 1 is anon-limiting example of a quantitative microscopy assembly. Otherexamples may include variations in quantities of individual components,such as a number of dichroic, excitation, and emission filters, aconfiguration of the light source, relative positioning of thecomponents, etc. Specific examples of how the quantitative microscopyassembly may be arranged to increase throughput while providing highresolution results is described herein.

In one example, the quantitative microscopy assembly, e.g., the assembly100 of FIG. 1 , may be used for high frequency screening of biologicalsamples. The microplate, supporting a plurality of specimens arranged inan array of wells of the microplate, may be positioned in a FOV of adetector of the assembly. Due to an inverse relationship between a sizeof the FOV and a magnification provided by the objective, the assemblymay only obtain a high resolution image of a portion of the microplatewith each image acquisition. In order to collect a complete data set ofthe plurality of samples, numerous image acquisitions may be demanded,delaying processing of the images and generation of analytical results.For applications such as drug screening, such delays may be costly.

In order to expedite image collection, a multi-detector quantitativemicroscopy system (hereafter, multi-detector system) may be used torapidly obtain high resolution images of the microplate samples, therebyenabling faster screening. In particular, faster screening of a singlemicroplate is enabled which provides increased resolution oftime-dependent biological events for large screenings. Additionally,when applied to screening of live cells, more microplates may beprocessing within a smaller time period during which sample integrity ispreserved. For kinetic assays, cycle times are faster, allowing themicroplates to be analyzed multiple times.

A configuration of the multi-detector system may be influenced bydimensions of the microplate which affects a positioning of a pluralityof objectives of the multi-detector system. For example, a spacingbetween the plurality of objectives may be maintained within a targetdistance to optimize respective overlapping of FOVs to generate acohesive, continuous, and complete image. Furthermore, the spacing ofthe plurality of objectives allows each detector of the multi-detectorto be actively collecting images during every imaging event. In someexamples, the detectors may be configured to obtain different types ofdata, thus expanding a capability of the multi-detector system tocapture new and unprecedented information. An arrangement of remainingcomponents of the multi-detector system may be thus configured toaccommodate the spacing and orientations of the plurality of objectivesrelative to the microplate. Furthermore, it may be desirable to maintaina footprint of the multi-detector system as small as possible.

In one example, as shown in FIG. 2 , a multi-detector system 200 may beformed of four individual blades 202 arranged in an x-shapedconfiguration. The multi-detector system 200 is depicted in FIG. 2 withan upper portion of the system and portions of a housing 220 of themulti-detector system 200 omitted for clarity. The upper portion mayinclude a top plate of the housing 220 of the multi-detector system 200as well as a sample receiving assembly. A set of reference axes 201 isprovided, indicating a y-axis, an x-axis, and a z-axis. In one example,the z-axis may be parallel with a direction of gravity. Furthermore, acentral axis 204 of the multi-detector system 200 may be parallel withthe z-axis. Each blade 202 of the multi-detector system 200 may includeat least the components depicted in the assembly 100 of FIG. 1 and theblades 202 may be operated concurrently to collect image data inparallel. Each blade 202 therefore forms an individual quantitativemicroscopy assembly. The components arranged in each of the blades 202may be positioned to optimize both an FOV of an objective of each bladeand a magnification/resolution of the resulting images. As such, thecomponents may be arranged in a vertical orientation, e.g., as a stackalong each blade.

For example, each blade 202 of the multi-detector system 200 may besimilarly configured, including a vertically oriented plate 206supporting a variety of components. An objective 208, which may be anembodiment of the objective 118 of FIG. 1 , may be positioned at a topof each blade 202 with other components of each blade 202 arranged belowthe objective 208, with respect to the z-axis. The blade 202 may have afront side 210 and a back side 212 and the front side 210 of the blade202 is depicted in greater detail in FIG. 3 .

The multi-detector system 200 may offer several advantageous overconventional systems. For example, conventional systems may employmultiple detectors to enable parallel imaging of microplates, therebyincreasing throughput. However, the conventional systems may not enablea higher imaging frequency of a single microplate. As a result of apackaging of the multi-detector system 200, and in particular, anarrangement of the objectives 118 at an upper portion of themulti-detector system 200, below the microplate, the quantitativemicroscopy assemblies of the multi-detector system 200 may synchronouslycapture images of portions of the microplate. The images may be combinedto form a complete image of the microplate, thus expediting a speed atwhich each well of the microplate is indexed. The packaging of themulti-detector system 200 allows the multi-detector system 200 to have asimilar footprint to a system with only one quantitative microscopyassembly.

Furthermore, in one example, the high speed imaging provided by themulti-detector system 200 may allow the multi-detector system 200 to beused for imaging live biological specimens in addition to endpointassays. In addition to the imaging speed, a fast framerate and highcycling frequency of the multi-detector system 200 enables newobservations of biology, such as live events and transient cell signals,which may otherwise be challenging to obtain using the conventionalsystems. As a result, cellular models may be constructed with greateraccuracy.

In addition, the arrangement of the objectives enables both high speedimaging of the microplate and efficient packaging of other components ofthe quantitative microscopy assemblies, such as a detector and a lightsource, to minimize the footprint of the multi-detector system 200. Theobjectives may be positioned relative to one another with a targetdistancing or spacing therebetween that accommodates a specific geometryof the microplate and reduces instances where a focus of any of theobjectives migrates outside of a target imaging region of themicroplate. A resulting capability of the objectives to rapidly captureimages of the microplate in a synchronized manner may not be readilyreplicated with other arrangements of the objectives.

In some examples, the multi-detector system 200 may be furtherconfigured with environmental control capabilities. For example, themicroplate may be enclosed within a sealed structure such that exposureof the sample to temperature, humidity, carbon dioxide level, etc., maybe regulated. Furthermore, the multi-detector system 200 may be adaptedwith a rapid, automated microplate changing mechanism, such as anautomated robotic arm.

Turning now to a view, as shown in FIG. 3 , of the front side 210 of theblade 202, the blade 202 has two vertical sides, e.g., parallel with thez-axis, including an inner side 302, e.g., proximate to the central axis204 as shown in FIG. 2 , and an outer side 304 that is distal to thecentral axis 204. The blade 202 may have an upper region 306, a middleregion 308 and a lower region 310, the upper region 306 positioned abovethe middle region 308 and the middle region 308 positioned above thelower region 310, along the z-axis. Each region may form a similarportion of a height 312, as defined along the z-axis, of the blade 202.The blade 202 may have a generally rectangular geometry with a width 314that is smaller, e.g., of a lesser distance, than the height 312.

As described above, the blade 202 may be formed of a plate 206 which isa structural support to which a variety of components may be coupled.The plate 206 may therefore be formed of a rigid, durable material suchas aluminum, another type of metal, plastic, etc. Edges of the plate 206may include sections configured to support specific components of thequantitative microscopy assembly and of the multi-detector system 200.For example, the plate 206 may include a notched section 316 along theinner side 302 of the blade 202 in the lower region 310 such that thewidth 314 of the blade 202 at the lower region 310 is narrower thatalong the middle region 308 and the upper region 306.

Furthermore, the plate 206 may include a protrusion 318 extendingupwards, with respect to the z-axis, and positioned at a mid-regionalong the width 314 of the blade 202. The plate 206 may be narrowest atthe protrusion 318. The protrusion 318 may be generally triangular witha sloped edge 320 (e.g., sloped relative to the y-axis), and a straightedge 322 that is parallel with the z-axis. Various casings and coversmay be coupled to the plate 206 via fasteners, such as screws, bolts,etc., to shield and cover components of the quantitative microscopyassembly.

At the upper region 306 of the blade 202, the objective 208 may beattached to the plate 206 along the inner side 302 such that theobjective 208 is aligned parallel with an inner edge 324 of the plate206 at the upper region 306 but protrudes beyond the inner edge 324 inan inwards direction (e.g., towards the central axis 204 shown in FIG. 2) along the x-axis. As such, the objective 208 may be located at anupper left-hand corner (when viewing the front side 210 of the blade202) of the blade 202 and may also protrude above a top edge 326 of theplate 206 at the protrusion 318. Furthermore, the objective 208 may beadjustable to translate along the z-axis to allow focusing of an imageprojected by the objective 208. Translation of a position of theobjective 208 along the z-axis may be enabled by a motor, e.g., asincluded in an objective module 328 and described further below, or maybe adjusted manually.

The objective 208 may be a substantially cylindrical component formed ofa plurality of lenses enclosed within a barrel, the plurality of lensesconfigured to provide a target magnification of an image. The objective208 may thus have a specific magnification and numerical aperture (NA),where the NA is a value indicating a range of angles over which theobjective 208 can accept or emit light. The objective 208 is a componentof the quantitative microscopy assembly positioned closest to (andbelow) a sample and may gather light from the sample and focus the lightto produce an image. As magnification provided by the objective 208increases, a field-of-view (FOV) of the objective 208 decreases. In oneexample, the objective 208 may include fixed focus lenses and may betherefore used at a specific working distance, such as a distancebetween 0.5-2 mm.

The objective 208 may be attached to the plate 206 by the objectivemodule 328 extending between the objective and the protrusion 318 of theplate 206. The objective module may include a bracket fixedly couplingthe objective 208 to the objective module 328, an objective mover whichmay be a motor that adjusts the position of the objective 208 along thez-axis, as well as a position sensor to monitor the position of theobjective along the z-axis. The objective mover may be controlled, e.g.,activated/deactivated, by a controller such as controller 124 of FIG. 1. The objective module 328 may be attached to the plate 206 by aplurality of fasteners 330 which may also be used to secure othercomponents to the plate 206. A first optical passage 332 may extendvertically (e.g., along the z-axis) directly below the objective 208 andmay have a cylindrical housing coupled to the plate 206, also via theplurality of fasteners 330. The first optical passage 332 may enclosemicroscope components, described further below with reference to FIG. 4, and may be an embodiment of the filter block 106 of FIG. 1 .

The first optical passage 332 may extend from a midpoint from below theobjective 208 in the upper region 306 of the blade 202 to a mid-pointalong the height 312 of the blade 202 in the middle region 308. An outerdiameter 334 of the first optical passage 332 may be larger than anouter diameter 336 of the objective 208. A camera 338, which may be anembodiment of the detector 122 of FIG. 1 , may be coupled to a bottomend 340 of the first optical passage 332.

In one example, the camera 338 may be a CCD camera configured to convertan electrical signal into optical image or video using a CCD. In anotherexample, the camera 338 may be a complementary metal oxide semiconductor(CMOS) camera which utilizes metal oxide semiconductors to convert lightinto electrical signals. The camera 338 may be positioned to receiveemission light from an illuminated sample to allow analysis of an imagegenerated by the camera 338, the emission light delivered from thesample to the camera 338 via the objective 208 and the first opticalpassage 332. The camera 338 is thereby optically coupled to theobjective 208 by the first optical passage 332. The camera 338 may bemounted at the bottom end 340 of the first optical passage 332 andmaintained in place by a bracket or some other supporting mechanism.

The plate 206 of the blade 202 may also support a laser auto-focus (LAF)sensor 342 arranged proximate to the outer side 304 of the blade 202.The LAF sensor 342 may be enclosed in a rectangular cover secured to theplate 206 by fastening devices, such as the plurality of fasteners 330or some other fastening mechanism. The LAF sensor 342 may emit a laserbeam into an optical path of the quantitative microscopy assembly, suchthat the LAF sensor 342 may use the laser beam as an optical probe todetermine a focus of the quantitative microscopy assembly. In oneexample, the LAF sensor 342 may be configured to produce a 785 nm redlaser beam from a light source such as a laser diode. The laser beam maybe reflected from a surface of the sample or a surface of a sampleholder (e.g., a microplate) and return to the LAF sensor 342 as anoptical signal which may be used to assess a focus of the quantitativemicroscopy assembly.

The LAF sensor 342 may be positioned such that a longest dimension ofthe LAF sensor 342, e.g., a length 339 of the LAF sensor 342, isparallel with the height 312 of the blade 202. By positioning the LAFsensor 342 as shown in FIGS. 3 and 4 , a footprint of the blade 202 ismaintained small. Furthermore, the positioning of the LAF sensor 342enables a geometry of an optical path, e.g., the first optical passage332 and a second optical passage 346, described below, to be maintainedsmall, e.g., a length of each passage is configured to be as short aspossible.

The LAF sensor 342 may be oriented parallel with and spaced away fromthe first optical passage 332, extending along a portion of the height312 of the plate 206, between a mid-point along the upper region 306 anda mid-point along the middle region 308. A plurality of optical elementsmay be included in the LAF sensor 342, including a light source forgenerating the laser beam and a detector for receiving the reflectedlaser beam which may be processed by an LAF controller (describedbelow). In one example, the detector may be configured as a CCD or aCMOS detector. The plurality of optical elements may further include afocal plane array, one or more lenses, an aperture stop, a beamsplitter, etc. The laser beam may thereby be directed and shaped by theplurality of optical elements to generate the optical signal used toalign the focus of the quantitative microscopy assembly.

The LAF sensor 342 may transmit the laser beam at a top end 344 of theLAF sensor 342 in a direction perpendicular to an optical path of thefirst optical passage 332. For example, the second optical passage 346may extend horizontally, e.g., along the x-axis, between the top end 344of the LAF sensor 342 and the first optical passage 332, and may mergewith the first optical passage 332 at an intersection 333. The secondoptical passage 346 may be enclosed in a casing with flat surfaces, thecasing attached to the plate 206 by the plurality of fasteners 330 orother fastening devices and may merge continuously with the firstoptical passage 332 such that the first and second optical passages 332,346 are optically coupled. The laser beam emitted by the LAF sensor 342may thereby pass through the second optical passage 346 and merge withexcitation light. As the laser beam passes through the second opticalpassage 346, the laser beam may interact with components enclosed withinthe first and second optical passages 332, 346 that facilitatesdeflection and transmission of the laser beam and excitation light, asdescribed further below.

A geometry and positioning of the first optical passage 332 and thesecond optical passage 346 may allow the optical passages to be shorterin length than a conventional system (e.g., a system without verticalarrangement of microscope assemblies). An overall optical path of eachblade 202 of the multi-detector system 200 is therefore miniaturized,allowing the assembly to have smaller packaging demands and increasingan efficiency of incident light transmission to a sample.

The blade 202 may also include a light source 348, which may be anembodiment of the light source 102 of FIG. 1 , enclosed by a cover 349that is secured to the plate 206 by the plurality of fasteners 330.Details of the light source 348 are shown in FIG. 4 and describedfurther below. The cover 349 of the light source 102 may occupy aportion of the width 314 of the blade 202 similar to a distance betweenthe first optical passage 332 and the LAF sensor 342, and the cover 349of the light source 102 may have a rectangular outer geometry. The lightsource 348 may be positioned in a central region of the plate 206 suchthat the light source 348 (and the cover 349) is spaced away from alledges of the plate 206. In one example, a first distance 350 between thetop edge 326 of the plate 206 and a top edge 352 of the cover 349 of thelight source 348 may be less than a second distance 354 between a bottomedge 356 of the cover 349 and a bottom edge 358 of the plate 206.However, in other examples, the first distance 350 and the seconddistance 354 may be similar or the first distance may be greater thanthe second distance 354.

An LAF controller 360 may be arranged below, e.g., with respect to thez-axis, the light source 348 and the LAF sensor 342. The LAF controller360 may be a rectangular structure and may include various electroniccomponents for signal processing, operation of the LAF sensor,monitoring a status of the LAF sensor, etc., and adapted with aconnector 362 to allow coupling of a cable to the LAF sensor 342. Thecable may enable communication between the LAF sensor and the LAFcontroller 360. The LAF controller 360 may include a plurality of portsalong an outer end 364 of the LAF controller 360 to allow the LAFcontroller 360 to be connected to, for example, a system controller,such as the controller 124 of FIG. 1 .

Connectivity between the LAF controller and the system controller mayallow a position of the objective 208 to be adjusted based on analignment of the quantitative microscope assembly focus with a targetfocal plane as detected by the LAF sensor 342. For example, when thefocus is determined to be out of alignment with the target focal plane,the LAF controller 360 may inform the system controller of an amount ofoffset of the focus from the target focal plane. In response to theinformation from the LAF controller 360, the system controller maycommand adjustment of the objective 208 by activating the objectivemover of the objective module 328 and modifying the position of theobjective 208 along the z-axis accordingly.

The LAF controller 360 may be mounted onto brackets which allow the LAFcontroller 360 to be secured to the plate 206 by the plurality offasteners 330. The outer end 364 of the LAF controller 360 may bealigned and flush with the outer side 304 of the blade 202 (and of theplate 206) and an inner end 366 of the LAF controller 360 may be spacedaway from the inner edge 324 of the plate 206. An upper side 368 of theLAF controller 360 may be spaced away from the bottom edge 356 of thecover 349 of the light source 348 and a bottom side 370 of the LAFcontroller 360 may be spaced away from the bottom edge 358 of the plate206.

The plate 206 may further include a plurality of apertures 372 arrangedbetween the LAF controller 360 and the bottom edge 358 of the plate 206.As shown in FIG. 3 , the plurality of apertures 372 may be aligned alongthe axis and separated into two groups, each with three apertures.Fasteners may be inserted through the plurality of apertures 372 toattached the plate 206 to a mounting device, such as a bracket, used tocouple the plate 206 to a base, e.g., the base 222 of FIG. 2 , of themulti-detector system 200.

A region of the blade 202, indicated by a dashed rectangle 374 in FIG. 3, is illustrated in FIG. 4 with select covers, housings, and casingsremoved to reveal shielded inner components. For example, as depicted inFIG. 4 , the cover 349 (e.g., as shown in FIG. 3 ) of the light source348 is removed, as is the housing of the first optical passage 332 andthe casing of the second optical passage 346. The plate 206 is omittedin FIG. 4 for brevity. Turning first to the first optical passage 332,an emission filter 402 may be arranged in the first optical passage 332proximate to the intersection 333 of the first optical passage 332 andthe second optical passage 346. The emission filter 402 may be anexample of the emission filter 114 of FIG. 1 , configured to removeundesired wavelengths from emission light travelling from the objective208 to the camera 338. Materials from which the emission filter 402 maybe formed include colored glass, glass with a dielectric optical coatingfor a specific wavelength, acrylic, etc., and may be configured withlongpass or bandpass transmission.

The emission filter 402 may be directly coupled to a tube lens 404 suchthat light filtered by the emission filter 402 immediately passesthrough the tube lens 404. In one example, as shown in FIG. 4 , theemission filter 402 may be attached to the tube lens 404 to form asingle unit. For example, the emission filter 402 may include a framewith a fitting configured to have a press-fit connection or threadedconnection to mate with a similar connection at the tube lens 404.

The tube lens 404 may have optical properties configured to complementoptical properties of the objective 208. The tube lens 404 is separatedand spaced away from the objective 208 by a distance 406. In oneexample, the distance 406 may be between 50-200 mm. In a second example,the distance 406 may be between 85-90 mm. However, other distances arepossible. The tube lens 404 may be decoupled from the objective 208 toallow variable pairing of tube lens focal length with objectivemagnification to achieve a desired balance between resolution and FOV.For example, a conventional system may utilize an objective 208 with a20× magnification in conjunction with a 200 mm focal length tube lens.In the quantitative microscopy assembly described herein, the tube lens404 may instead have a 100 mm focal length and, when combined with the20× magnification provided by the objective 208, allows the quantitativemicroscopy assembly to have both a larger FOV and higher resolution thanthe conventional system. The decoupled pairing of the objective 208 andthe tube lens 404 may contribute at least partially to a higherthroughput of the multi-detector system by increasing the FOV of eachobjective of the system and thereby generating an image from a largersection of a microplate while capturing fine details within the imagedportion.

A bottom end 407 of the tube lens 404 may be connected to a camera mount408 which couples the camera 338 to the tube lens 404 and maintains theposition of the camera 338. For example, camera 338 may have a threadedengagement with the camera mount 408. The camera mount 408 may besimilarly coupled to the tube lens 404 and may have a length 410configured to dissipate heat between the tube lens 404 and the camera338. As such, the camera mount 408 may also be a thermal isolator andmay assist in thermal management at the camera 338. For example, anyheat generated by interaction of the emission light with the emissionfilter 402 and/or tube lens 404 may be absorbed by the camera mount 408.

The camera mount 408 may be formed of a plastic with insulatingproperties. By attaching the camera 338 to the first optical passage 332by way of the camera mount 408, the camera 338 does not directly contactmetallic components of the quantitative microscopy assembly. Forexample, the camera 338 does not contact the housing of the firstoptical passage 332, or the tube lens 404, which may be formed of orinclude parts formed of a metal such as aluminum. Furthermore, thecamera is spaced away from the plate 206 and the cover 349 (as shown inFIG. 3 ) of the light source 348, which may also be formed of a metalsuch as aluminum, stainless steel, etc. The camera 338 is thereforeisolated from heat conducting materials of the quantitative microscopyassembly.

The light source 348 includes light-emitting diodes (LEDs) 412 arrangedaround a set of dichroic mirrors 414. The LEDs includes a first LED 412a, a second LED 412 b, a third LED 412 c, and a fourth LED 412 d. Thefirst and second LEDs 412 a, 412 b may be adjacent to one another andaligned along the x-axis and positioned proximate to a lower (withrespect to the z-axis) end of the light source 348. The first LED 412 amay be closer to the camera 338 than the second LED 412 b. The third LED412 c and the fourth LED 412 d may be aligned along the z-axis andpositioned adjacent to one another along a side of the light source 348proximate to the LAF sensor 342. The fourth LED 412 d may be arrangedabove the third LED 412 c. The first and second LEDs 412 a, 412 b form afirst group of the LEDs that is oriented perpendicular to a second groupof the LEDs, formed of the third and fourth LEDs 412 c, 412 d.

Each of the LEDs 412 may be configured to emit light of differentwavelengths. For example, the first LED 412 a may have a centerwavelength of 630 nm, the second LED 412 b may have a center wavelengthof 470 nm, the third LED 412 c may have a center wavelength of 390 nm,and the fourth LED 412 d may have a center wavelength of 555 nm.However, other center wavelengths may be possible. During sampleimaging, each of the LEDs 412 may be individually activated toilluminate the sample with different wavelengths of light, according towhich of the LEDs 412 is activated. A separate image of the sample maybe obtained from each illumination channel of the LEDs 412 whichprovides images depicting variations in sample fluorescence depending onthe center wavelength of the incident light.

By directly coupling the light source 348 to the blade 202, rather thanpositioning the light source external to the blade 202 and distal toother imaging components supported on the plate 206, an illuminationprovided by the light source 348 may be brighter. As a result, shorterexposure times are enabled which may lead to increased imaging speed. Inother examples, the light source 348 may instead be coupled by anoptical cable, fiber optics, etc., but, as such, may provide less brightillumination.

The set of dichroic mirrors 414 may be arranged in an area between thefirst and second groups of the LEDs and includes a first dichroic mirror414 a, a second dichroic mirror 414 b, and a third dichroic mirror 414c. The first and second dichroic mirrors 414 a, 414 b may be alignedalong a common axis that is angled relative to the alignment of thefirst group of the LEDs 412 as well as to the alignment of the secondgroup of the LEDs 412. The third dichroic mirror 414 c is oriented alonga parallel but separate axis from the first and second dichroic mirrors414 a, 414 b such that the third dichroic mirror 414 c is offset fromthe first and second dichroic mirrors 414 a, 414 b and also centeredrelative to the first and second dichroic mirrors 414 a, 414 b. The setof dichroic mirrors 414 are spaced away from each of the LEDs 412 andoriented at an angle a relative to the x-axis. In one example, α may be45 degrees.

The location and angle of the set of dichroic mirrors 414 relative tothe LEDs 412 may be configured to allow a target set of wavelengths tobe transmitted to the sample while reflecting shorter wavelengths. Eachof the set of dichroic mirrors 414 may be long-pass (LP) dichroic mirrorconfigured with a specific wavelength threshold above which light withsufficiently long wavelengths is able to pass therethrough. The set ofdichroic mirrors 414 may therefore be arranged such that each of the setof dichroic mirrors 414 is positioned in a pathway of a suitable LED ofthe LEDs 412. Light generated at each of the LEDs 412 interacts with atleast one of the dichroic mirrors 414 before passing through an outputlight guide 416.

The transmitted/reflected light (e.g., excitation light) from each ofthe LEDs 412 may be reflected from the first dichroic mirror 414 a,through the output light guide 416 along a linear path 418 a parallelwith the z-axis, to a first selective mirror 420. A top of the outputlight guide 416 may be aligned with a top of the emission filter 402along the x-axis, for example. The first selective mirror 420 may bepositioned in the second optical path 346 and oriented at a similarangle with respect to the x-axis as the angle a of the set of dichroicmirrors 414, e.g., 45 degrees.

The excitation light from the LEDs 412 may be reflected at the firstselective mirror 420 by 90 degrees, as indicated by arrow 422. Thereflected excitation light travels parallel to the x-axis in the secondoptical path 346 from the first selective mirror 420 to a secondselective mirror 424. The first selective mirror 420 may be configuredwith a coating that causes light of wavelengths below a thresholdwavelength to be reflected while light above the threshold wavelengthmay be transmitted without interference or obstruction through the firstselective mirror 420. For example, the threshold wavelength of the firstselective mirror 420 may be 700 nm. All excitation light from the LEDs412 are therefore reflected at the first selective mirror 420 while thelaser beam (e.g., the 785 nm red laser) from the LAF sensor 342 passesthrough the first selective mirror 420, as indicated by arrow 426.

At the second selective mirror 424, the excitation light and a firstportion of the laser beam are reflected by 90 degrees, and merge along acommon, linear path upwards along the z-axis, as indicated by arrow 428,through the first optical passage 332 to the objective 208. A beamformed of the excitation light and the first portion of the laser beammay continue through the objective to the sample where the excitationlight induces fluorescence of the sample. The second selective mirror424 may be arranged in the first optical passage 332 at the intersection333 of the first optical passage 332 with the second optical passage346. The second selective mirror 424 may be oriented at a similar anglerelative to the x-axis as the first selective mirror 420, e.g., at theangle a of the set of dichroic mirrors 414. The first portion of thelaser beam reflected at the second selective mirror 424 may representmost of the laser beam, e.g., more than 50% of the laser beam photons.The second portion of the laser beam is smaller than the first portionand may be transmitted through the second selective mirror 424 maycontinue along the x-axis to be attenuated at the housing of the firstoptical passage 332. The LAF sensor 342 may be preferentially tuned tominimize the second portion of the laser beam.

Upon illumination by the excitation light, the sample may emit light ata different wavelength than a wavelength that induces fluorescence. Theemitted light, e.g., emission light, may travel along a linear path fromthe sample to the camera 338 through the first optical passage 332, asindicated by arrow 430. As such, the second selective mirror 424 ispositioned in a path of the emission light and may be configured toallow the emission light to pass therethrough unobstructed. The secondselective mirror 424 may thus be adapted with a coating that reflectswavelengths generated by the LEDs 412 and the LAF sensor 342 buttransmits expected wavelengths of the emission light. For example, thesecond selective mirror 424 may allow transmission of wavelengthsbetween 400 and 700 nm. The transmitted light is then filtered as itpasses through the emission filter 402, as described above.

The light source 348 may be powered and controlled by a printed circuitboard assembly (PCBA). An example of a PCBA 502 is depicted in FIG. 5 ,which may be coupled to the back side 212 of the blade 202, as shown inFIG. 2 . The PCBA 502 is shown without the plate 206 and othercomponents coupled to the plate 206 with the exception of the tube lens404, the camera mount 408, and the output light guide 416. The PCBA 502may include various electronic components coupled to a PCB 504. Forexample, the LEDs 412 of FIG. 4 may be directly coupled to the PCB 504via soldering. The various electronic components may further includediodes, capacitors, resistors, switches, inductors, etc. The positioningof the PCBA 502 on the back side of the blade 202, opposite of the lightsource 348, further contributes to maintaining a small footprint of eachblade 202 of the multi-detector system 200 of FIG. 2 .

A packaging of the components of the quantitative microscopy assemblyillustrated in FIGS. 3-5 may provide an optimized balance betweensuitable distancing between components and minimizing overall dimensionsof the blade to accommodate screening of the sample, e.g., distributedin wells of a microplate, above the blades. For example, a distancebetween a seat of the objective and the tube lens may be no more than100 mm. An optimal distance between the seat of the objective and theLAF sensor may be 155 mm and an optimal distance between the seat of theobjective and the light source may be 190 mm. By utilizing thearrangement of the blade shown in FIGS. 3 and 4 , performances of thecomponents may be maintained high without incurring wasted space, e.g.,unoccupied and non-useful regions of the blade.

As described above, the vertical stacking of the components of eachblade of the multi-detector system 200 of FIG. 2 enables imaging of amicroplate arranged above the blades 202. An example of a microplate 600which may be coupled to a plate holder of the multi-detector system 200is illustrated in FIG. 6 . The microplate 600 has a rectangular outergeometry with a length 604 that is greater than a width 606 of themicroplate 600 and includes sidewalls 608 surrounding an array of wells610. The length 604 and the width 606 of the microplate 600 may conformto specific dimensions according to target specifications, such asANSI/SLAS specifications. As an example, the length 604 of themicroplate 600 may be 127.76 mm and the width 606 of the microplate 600may be 85.48 mm.

In one example, the microplate 600 may have 96 of the wells 610distributed evenly (e.g., evenly spaced apart) across the microplate600. The wells 610 may each have a depth, e.g., along the z-axis,between 10-20 mm and a volume of up to 400 μL, for example, althoughother depths and volumes are possible. Physical characteristics of themicroplate 600 may vary according to a type of applied analysis. Forexample, when the microplate 600 is used for fluorescence imaging, themicroplate 600 may be configured with black walls, e.g., walls of thewells and the sidewalls 608 are black, to reduce backgroundautofluorescence when short half-life fluorophores are used. However,when long half-life fluorophores are used, the walls of the wells 610and the sidewalls 608 of the microplate 600 may be white to increasefluorescent signals. Floors of the wells 610 may be formed of a clearmaterial to allow imaging of the wells 610 from below the microplate600.

The microplate 600 may have a foot 612 that forms a base platform of themicroplate 600. The foot 612 extends around a perimeter of themicroplate 600 at a bottom of the microplate 600 (relative to thez-axis), and extends beyond the sidewalls 608 in the y-x plane. The foot612 may form a smaller portion of a height 614 of the microplate 600than the sidewalls 608.

The sidewalls 608 may include at least one chamfer 616. The microplate600 of FIGS. 6-7 and 9-10 includes two chamfers 616. Other examples ofthe microplate may include a single chamfer, chamfers at eachintersection of the side walls, no chamfers, etc. When the microplate600 is used to support samples for imaging at the multi-detector system200, a position of the microplate 600 above the blades 202 of themulti-detector system 200 may be maintained by an assembly configured tocontact the microplate 600 and impede undesired movement of themicroplate 600.

An example of a plate holder 700 for the multi-detector system 200 isdepicted in FIGS. 7-10 . The plate holder 700 is illustrated from a topview with the microplate 600 in FIG. 7 and from the top view but withoutthe microplate 600 in FIG. 8 . A section of the plate holder 700 isshown in FIG. 9 , also from the top view, and the plate holder isdepicted from a bottom view in FIG. 10 . Turning first to FIG. 7 , theplate holder includes a frame 702, a swing arm 704, an extension spring706, a whippletree assembly 708, a swing arm pivot 710, and a bearing712.

The frame 702 may have an irregular (e.g., asymmetric along each of they-z plane and the z-x plane) outer geometry and includes an opening 714.The opening 714 is shown in FIG. 8 without the microplate 600, which maybe positioned over the opening 714, illustrating a symmetric shape ofthe opening 714 across the y-z plane and the z-x plane. A first, maximumlength 802 of the opening 714 may be greater than a first, maximum width804 of the opening 714. Moreover, the first length 802 of the opening714 may be greater than the length 604 of the microplate 600 (as shownin FIG. 6 ) and the first width 804 of the opening 714 may be greaterthe width 606 of the microplate 600 (as shown in FIG. 6 ).

A portion of the frame 702 surrounding the opening 714 has a firststepped region 806 which may form four corners around a perimeter of theopening 714. The first stepped region 806 may further form a portion ofthe opening 714 with a second length 808 that is shorter than the firstlength 802 of the opening 714 and similar to the length 604 of themicroplate 600. Additionally, the opening 714 may include a secondstepped region 810 also forming four corners around the perimeter of theopening 714, the four corners of the second stepped region 810 offsetfrom the four corners of the first stepped region 806. The secondstepped region 810 may form a portion of opening 714 with a second width812 that is narrower than the first width 804 and similar to the width606 of the microplate 600.

The opening 714 further includes pads or braces 814 corresponding tocorners of the microplate 600 when the microplate 600 is positioned andcentered in the opening 714. The braces 814 may be arranged under themicroplate 600 to support a vertical position (e.g., with respect to thez-axis) of the microplate 600 and may be in contact with a bottomsurface of the microplate 600 at the corners of the foot 612 of themicroplate 600 to support a weight of the microplate 600. Verticalsurfaces 816 of the frame 702 coupled to the braces 814 may thereforeprovide guides against which the corners of the microplate 600 may abut.When the microplate 600 is placed in the opening 714, the microplate 600is supported by the frame 702 with gaps in the frame extending alongeach side of the microplate 600, as shown in FIG. 7 . The gaps may besufficiently large to allow a user's fingers to inserted therethrough,allowing the microplate 600 to be easily placed onto the plate holder700 without unintentional bumping of user's fingers against the frame702.

The swing arm 704 may extend along a portion of the frame 702 adjacentto the opening 714. For example, as shown in FIGS. 7-10 , the swing arm704 may be located to the left of the opening in the plate holder 700. Alength 720 of the swing arm may be greater than the width 606 of themicroplate 600 but shorter than a maximum width 722 of the frame 702. Asshown in FIG. 7 , the swing arm 704 may include a first portion 716 anda second portion 718 which may be continuous with one another. The firstportion 716 may extend along the frame 702 linearly and parallel withthe y-axis. The second portion 718 may also extend linearly along theframe 702 but at an angle β relative to the y-axis. In one example, theangle β may be 45 degrees. In other examples, however, the angle β maybe an angle between 30 to 60 degrees.

The angled extension of the second portion 718 of the swing arm 704allows a head 724 of the swing arm 704 to be positioned adjacent to themicroplate 600 at a top left-hand corner of the microplate 600, withrespect to the top view depicted in FIGS. 7-9 . The head 724 includes a90-degree cutout 744 to accommodate both an arrangement of thewhippletree assembly 708 thereat and a protrusion of a first corner 746of the microplate 600 into the head 724 of the swing arm 704. The90-degree cutout 744 is located along an edge of the swing arm 704proximate to the microplate 600. The extension spring 706 may be coupledto a first end 726 of the swing arm 704, the first end 726 included inthe head 724 of the swing arm 704. The extension spring 706 may beattached at one end to the swing arm 704 by a first fastener 728, suchas a bolt, and attached at an opposite end to the frame 702 by a similarsecond fastener 730.

The extension spring 706 may extend between the first and secondfasteners 728, 730 parallel with the x-axis and is depicted in a first,stretched position in FIGS. 7 and 8 . In other words, the extensionspring 706 may be under tension due to a stiffness of the extensionspring 706 that resists stretching. For example, the extension spring706 may be configured with a high stiffness, e.g., a high resistance toelongation of the extension spring 706. As an example, the extensionspring 706 may have a spring force of 1.3-2.3 pounds.

The whippletree assembly 708 may be positioned at the head 724 of theswing arm 704 and aligned with a diagonal axis of the microplate 600.The whippletree assembly 708 may include a pivot 732 and a yoke 734which may be illustrated with greater clarity in an expanded view 900 ofa section of the plate holder 700 indicated by dashed area 736 and shownin FIG. 9 . The expanded view 900 of FIG. 9 shows the whippletreeassembly 708 with the swing arm 704 omitted.

The yoke 734 may be a bracket with two arms 902 oriented perpendicularto one another, including a first arm 902 a extending parallel with thex-axis and a second arm 902 b extending parallel with the y-axis. Theyoke 734 is coupled to the frame 702 of the plate holder 700 at anintersection of the arms 902 by the pivot 732. In addition to securingthe yoke 734 to the frame 702, the pivot 732 may also control a degreeto which the yoke 734 may rotate around the pivot 732, as indicated byarrow 904.

For example, the pivot 732 may include a spring mechanism thatconstrains rotation of the yoke 734 in either a clockwise orcounter-clockwise direction to a threshold degree of rotation. In oneexample, the threshold degree of rotation may be 10 degrees in eitherdirection relative to the position of the yoke 734 shown in FIG. 9 . Inother examples, the yoke 734 may pivot 1.25 degrees in either directionrelative to the position of the yoke 734 shown in FIG. 9 . In oneexample, the spring mechanism of the pivot 732 is excluded and thethreshold degree of rotation (e.g., pivoting of the yoke 734 1.25degrees in either direction) is established by hard stops in a range ofrotation of the yoke 732 established by geometry of the yoke 732. Theyoke 732 may pivot within the range of rotation. Resistance of the pivot732 to rotation of the yoke 734 beyond the threshold degree of rotationmay be equal in both directions. Furthermore, in examples including thespring mechanism, the yoke 734 may be maintained oriented such that thefirst arm 902 a remains parallel with the x-axis and the second arm 902b remains parallel with the y-axis regardless of a position of the swingarm 704.

A pressure exerted on the microplate 600 by the whippletree assembly 708may be evenly distributed between the arms 902 of the yoke 734 based ona combination of an angle of the swing arm 704 relative to themicroplate 600 and transmission of the stiffness of the extension spring706 to the microplate 600 as a compressive force. The arms 902 may exertpressure on the microplate 600 due to tension at the extension spring706 (described further below) via a first contact ball 906 coupled to anend of the first arm 902 a and a second contact ball 908 coupled to anend of the second arm 902 b.

The swing arm 704 may be configured to rotate about the swing arm pivot710 (described further below in detail) such that the head 724 swingstoward and away from the microplate 600, as indicated by arrow 748, atan angle y relative to a longitudinal axis 750 of the microplate 600, asshown in FIG. 7 . In one example, the angle γ is 45 degrees which allowsthe first and second contact balls 906, 908 to contact the microplate600 at the same time when the head 724 of the swing arm 704 travelstoward the microplate 600. The first and second contact balls 906, 908may be interfaces between the whippletree assembly 708 and themicroplate 600 and may press against the microplate 600 in perpendiculardirections. The contact balls may be formed of a rigid material, such asstainless steel or aluminum. As another example, the contact balls maybe formed of a more flexible material, such as rubber.

For example, when the whippletree assembly 708 presses against themicroplate, the first arm 902 a of the yoke 734 may exert a forceagainst a first section 910 of the sidewalls 608 of the microplate 600.The force is transmitted through the first contact ball 906, asindicated by arrow 912. The second arm 902 b of the yoke 734 may exert aforce against a second section 914, the second section 914 perpendicularto the first section 910, of the sidewalls 608 of the microplate 600.The force is transmitted through the second contact ball 908 asindicated by arrow 916 and may be equal to the force transmitted throughthe first contact ball 906. In this way, the forces applied to sidewalls608 of the microplate 600 from the whippletree assembly 708 aredistributed evenly between the first contact ball 906 and the secondcontact ball 908 in two directions normal to one another, therebystabilizing the position of the microplate 600 in the opening 714 of theframe 702. For example, as shown in FIG. 9 , the first arm 902 a of theyoke 734 may press the microplate 600 against the first stepped region806 as indicated by arrows 738 and the second arm 902 b of the yoke 734may press the microplate 600 against the first stepped region 806 asindicated by arrows 740.

By transmitting the force applied by the whippletree assembly 708 to themicroplate 600 through the first and second contact balls 906, 908, theforce is directed through a single, concentrated point where each of thefirst and the second contact balls 906, 908 directly contact thesidewalls 608 of the microplate 600, at points beyond, e.g., furtheralong the length 604 and the width 606 of the microplate 600 (as shownin FIG. 6 ), the chamfer 616 at the first corner 746. Application of theforce through the single, concentrated points of contact circumventstransmission of torque from the yoke 734, when the yoke 734 rotatesabout the pivot 732. In other words, regardless of how the yoke 734 isoriented relative to the pivot 732, the forces applied to the microplate600 from the yoke 734 remains evenly balanced between the first andsecond contact balls 906, 908 such that the microplate 600 is pressedagainst the frame 702 with a same compressive force in the directionindicated by arrows 738 as in the direction indicated by arrows 740, asshown in FIG. 7 .

In addition, by separating the points of contact between the whippletreeassembly 708 and the microplate 600, e.g., at the contact balls, thewhippletree assembly 708 may accommodate variations in microplate widthand length. Furthermore, an ability of the whippletree assembly 708 toclamp the microplate 600 in place is unchanged whether or not the firstcorner 746 of the microplate 600 includes the chamfer 616.

As shown in FIGS. 7 and 8 , the first portion 716 of the swing arm 704includes the swing arm pivot 710. The swing arm pivot 710 may be afulcrum about which the swing arm 704 may rotate, as indicated by arrow742 in FIG. 7 . In addition, the swing arm 704 may be connected to theframe 702 of the plate holder 700 by the swing arm pivot 710. In oneexample, the swing arm pivot 710 may be a bearing configured to allowpivoting of the swing arm 704 in either of a clockwise orcounter-clockwise direction. An amount of rotation of the swing arm 704in either direction may be constrained by the extension spring 706.

For example, as shown in FIG. 8 and indicated by arrow 818, when theswing arm 704 rotates in the counter-clockwise direction (with respectto the top view of the plate holder 700 shown in FIG. 8 ), the rotationis resisted by increased stretching of the extension spring 706 to asecond position. The resistance of the extension spring 706 to thecounter-clockwise rotation may increase as the swing arm 704 continuespivoting, which results in the head 724 of the swing arm 704 moving awayfrom the opening 714 of the frame 702, until the extension spring 706reaches a maximum stretching tolerance. When the extension spring is inthe second position, the contact balls of the yoke 734 are no longer incontact with the microplate 600. Tension on the extension spring 706increases as the head 724 swings farther away from the opening 714 ofthe frame (as well as from the first corner 746 of the microplate 600when the microplate 600 is positioned in the frame 702). As such, theswing arm 704 may rotate in the counter-clockwise direction to a maximumof five degrees in both directions of the orientation shown in FIG. 8without causing deformation of the extension spring 706.

Rotation of the swing arm 704 in the clockwise direction from theposition of the swing arm shown in FIGS. 7-10 may be experience lessresistance due to release of tension on the extension spring 706, e.g.,extension spring 706 becomes less tense, allowing the length of theextension spring 706 to decrease. When the extension spring 706 reachesthe first position, as shown in FIGS. 7 and 8 , the contact balls of theyoke 734 may press against the microplate 600 according to the stiffnessof the extension spring 706.

The spring force or stiffness of the extension spring 706, as well as alength of the extension spring (the length defined along the x-axis),may be configured to provide a suitable amount of tension when the firstand second contact balls 906, 908 of the whippletree assembly 708 are incontact with the microplate 600 when the microplate 600 is positioned inthe opening 714 of the frame 702 as shown in FIG. 7 . When themicroplate 600 is not present, the extension spring 706 may be in athird position where the extension spring 706 is at a minimum length andless stretched than in the first or second positions. The extensionspring 706 may be further stretched, such as 5-10% longer when thecontact balls engage with the microplate 600 compared to when themicroplate 600 is not present. Thus the counteracting stiffness of theextension spring 706 (e.g., resistance to stretching) may exert asuitable amount of compression on the microplate 600, as transmittedthrough the contact balls, to hold the microplate 600 securely withinthe opening 714 of the frame 702.

To release the microplate 600 from the force exerted by swing arm 704,the swing arm 704 may be pivoted about the swing arm pivot 710 in thecounter-clockwise direction until the force from the swing arm 704 isalleviated. In some examples, the swing arm 704 may be rotated until thecontact balls are no longer in contact with the microplate 600. Rotationof the swing arm 704 may be facilitated by adjustment of a position ofthe bearing 712.

For example, as shown in FIG. 10 in a bottom view of the plate holder700 with the frame 702 depicted as transparent for clarity, at least aportion of the bearing 712 is located under the frame 702. In otherwords, the bearing 712 may extend through a window 1002 of the frame 702with the portion of the bearing located under the frame 702 arranged onan opposite side of the frame 702 from the swing arm 704. The bearing712 may be coupled to the swing arm 704 through a window 1002 of theframe 702. In one example, the bearing 712 may be fixedly coupled to atail end 1004 of the swing arm 704.

The window 1002 is located directly below the tail end 1004 of the swingarm 704 and may have a greater width 1006 than a width of the swing arm704 at the tail end 1004. When the bearing 712 is shifted to the left,as indicated by arrow 1008, the swing arm 704 rotates about the swingarm pivot 710 as indicated by arrow 1010. As the swing arm 704 rotatesas indicated by arrow 1010, the head 724 swings away from the firstcorner 746 of the microplate 600, as indicated by arrow 1012, againstthe spring force of the extension spring 706. As described above withreference to FIG. 7 , the head 724 travels at the angle y relative tothe longitudinal axis 750 of the microplate 600. The forces exerted bythe first and the second contact balls 906, 908 of the whippletreeassembly 708 are removed from the microplate 600 at the same time whenthe head 724 moves away from the microplate 600. Movement of the bearing712 may be halted when the contact balls are no longer in contact withthe microplate 600.

When the contact balls of the whippletree assembly 708 are no longer incontact with the microplate 600, the microplate 600 may be removed,replaced, or added to the plate holder 700 without interference from theswing arm 704. To engage the swing arm 704 with the microplate 600,e.g., upon replacement or installation of the microplate 600 in theplate holder 700, the bearing 712 may be displaced, e.g., shifted, asindicated by arrow 1014, causing the swing arm 704 to pivot about theswing arm pivot 710 as indicated by arrow 1016. The head 724 of theswing arm 704 moves towards the first corner 746 of the microplate 600,as indicated by arrow 1018, until the contact balls come into contactwith the microplate 600, at which movement of the bearing 712 is halted.As described above, the extension spring 706 may remain under tension toclamp the microplate 600 within the opening 714 of the frame 702.

In one example, displacement of the bearing 712 may be controlled by anactuating system for the swing arm 704, including a motor 1102, a ram1104 with a ramped surface 1106, as shown in FIGS. 11A-11B in a top viewof the plate holder 700. It will be noted that the top view of FIGS.11A-11B is rotated by 90 degrees relative to the views of FIG. 7-9 . Theplate holder 700 is depicted coupled to a stage 1150 which adjusts aposition of the plate holder 700 along the x-y plane relative to themulti-detector system 200. The swing arm 704 is engaged with themicroplate 600 in FIG. 11A and disengaged from the microplate 600 inFIG. 11B.

The motor 1102 may be connected to the ram 1104, where the ram 1104includes the ramped surface 1106 protruding upwards (along the x-axis)from the ram 1104, and the ram 1104 may be configured to slide along aset of rods 1108, e.g., along the y-axis. The motor 1102 may be arrangedbelow a plane of the swing arm 704 (e.g., a y-x plane) and actuation ofthe motor 1102 may compel movement of the ram 1104 along the set of rods1108 which may control engagement of the swing arm 704 with themicroplate 600 via the position of the bearing 712, as described above.For example, when the swing arm 704 is engaged with the microplate 600,as shown in FIG. 11A, the bearing 712 is in a first position along thex-axis and the extension spring 706 is stretched to a first length 1110,corresponding to the first position of the extension spring 706. Whenstretched to the first length 1110, a corresponding amount of tension atthe extension spring 706 causes the first end 726 of the swing arm 704to apply a sufficient amount of compression, via the first and secondcontact balls 906, 908, to clamp the microplate 600 in place within theopening 714 of the frame 702 during translation of the plate holder 700by the stage 1150.

Furthermore, when the swing arm 704 is engaged, the ram 1104 isretracted to the right along the set of rods 1108, e.g., to a retractedposition. In the retracted position, the ram 1104 is positioned as closeto the motor 1102 as allowable by a first cross-beam 1107 coupling theset of rods 1108 to one another. When the ram 1104 abuts the firstcross-beam 1107, the ramped surface 1106 of the ram 1104 may be spacedaway from the bearing 712 by a maximum distance. When the motor 1102 isactuated to disengage the swing arm 704, the motor 1102 may drivesliding of the ram 1104 to the left, as indicated by arrow 1112 in FIG.11A. As the ram 1104 slides to the left, a distance between the rampedsurface 1106 and the bearing 712 decreases until the ramped surface 1106comes into contact with the bearing 712. Continued movement of the ram1104 to the left with the ramped surface 1106 in contact with thebearing 712 forces the bearing 712 to slide along the ramped surface1106. For example, the bearing 712 may move upwards, with respect to thex-axis, as indicated by arrow 1116.

The motor 1102 may continue propelling the ram 1104, against thestiffness of the extension spring 706, to the left until the ram 1104abuts a second cross-beam 1109 coupling the set of rods 1108 to oneanother, as shown in FIG. 11B. The ram 1104 is shifted to a fullyextended position in FIG. 11B and the displacement of the bearing 712upwards, along the x-axis causes the first end 726 of the swing arm 704to swing away from the microplate 600. The first and second contactballs 906, 908 are no longer in contact with the microplate 600. Theextension spring 706 is stretched further to a second length 1118 thatis greater than the first length 1110, thereby increasing tension at theextension spring 706.

When the ram 1104 is in the fully extended position of FIG. 11B, themicroplate 600 may be loaded, removed or replaced. In some examples,loading/removal/replacement may be performed manually, e.g., by a user,and in other examples, the loading/removal/replacement may be achievedby an automated system such as a robotic arm.

It will be appreciated that the actuating system forengaging/disengaging the swing arm 704 described above is a non-limitingexample and other mechanisms have been contemplated. For example, analternate mechanism may include using a non-moving surface and drivingthe bearing 712 to and from the non-moving surface using an actuationsystem of the stage 1150.

The microplate 600 may allow a large number of samples to be screenedconcurrently. By positioning the microplate 600 above the multi-detectorsystem 200, the blades 202 may synchronously capture imaging data fromportions of the microplate 600 to generate a complete image of themicroplate 600.

The blades 202 of the multi-detector system 200 may be configured asshown in FIGS. 3-5 to enable the x-shaped assembly of the multi-detectorsystem 200. The x-shaped configuration allows the objectives 208 of eachblade 202 to be positioned relative to one another with optimizedspacing in between. For example, as shown in FIG. 12 , the blades 202include (along a clockwise direction) a first blade 202 a with a firstobjective 208 a, a second blade 202 b with a second objective 208 b, athird blade 202 c with a third objective 208 c, and a fourth blade 202 dwith a fourth objective 208 d. The blades 202 are oriented such that theobjective 208 are proximate to a center of the x-shaped configurationand the LAF sensor 342 of each blade 202 is positioned at outer regionsof the x-shaped configuration.

Along a first axis 1202, the first axis 1202 parallel with the y-axis,the first objective 208 a is spaced away from the second objective 208 bby a first distance 1204. The third objective 208 c is also spaced awayfrom the fourth objective 208 d by the first distance 1204 along thefirst axis 1202. Along a second axis 1206, the second axis 1206 parallelwith the x-axis, the first objective 208 a is spaced away from thefourth objective 208 d by a second distance 1208. The second objective208 b is also spaced away from the third objective 208 c by the seconddistance 1208 along the second axis 1206.

The second distance 1208 is greater than the first distance 1204. In oneexample, the first distance may be 2.25 mm and the second distance 1208may be 20.57 mm. The differences in the distances may result in greateroverlap between FOVs of the objectives along the first axis 1202 thanthe second axis 1206. For example, the FOV of the first objective 208 amay overlap to a greater extent with the FOV of the second objective 208b than the fourth objective 208 d. Furthermore, the FOV of the firstobjective may overlap to a greater extent with the FOV of the fourthobjective 208 d than with the FOV of the third objective 208 c.

By incorporating a greater distance between the objectives 208 along thesecond axis 1206 than along the first axis 1202, an overall, combinedFOV of the objectives 208 may be larger along the second axis 1206 thanalong the first axis 1202. For example, a first FOV of the firstobjective 208 a is indicated by dashed circle 1210, a second FOV of thesecond objective 208 b is indicated by dashed circle 1212, a third FOVof the third objective 208 c is indicated by dashed circle 1214, and afourth FOV of the fourth objective 208 d is indicated by dashed circle1216. A combined, overall FOV resulting from an overlap between the FOVsof the objectives 208 has a width 1218 along the first axis 1202 and alength 1220 along the second axis 1206.

Due to the different spacing between the objectives 208 along the firstaxis 1202 versus the second axis 1206, the length 1220 is greater thanthe width 1218 of the overall FOV. As such, dimensions of the overallFOV accommodates the rectangular geometry of the microplate, e.g., themicroplate 600 of FIGS. 6-7 and 9-10 , as indicated by dashed rectangle1222. By spacing the objectives 208 apart by a greater distance alongthe length of microplate (e.g., along the x-axis), than the width of themicroplate (e.g., along the y-axis), a complete image capturing eachwell of the microplate may be obtained.

Furthermore, the spacing between the objectives 208, e.g., where thespacing is greater along the second axis 1206 than the first axis 1202where both the axes are horizontal axes arranged normal to one another,may enable efficient, high speed imaging of the microplate, e.g., themicroplate 600 of FIGS. 6 -, 7, and 9-11B. An arrangement of theobjective 208 results in a radial distribution of the objective FOVs,each configured with similarly large FOVs, such that each objectivecaptures a portion of the microplate with each imaging event and a focusof the objectives does not stray beyond, e.g., outside of, the imagingarea of the microplate, even when the microplate position is adjusted.An overall rectangular orientation of the objectives 208 enablescomplete imaging of the entire microplate simultaneously. Imaging of themicroplate may be repeatedly cycled at a high rate as a result.

Returning to FIG. 2 , the blades 202 of the multi-detector system 200may be coupled to an isolated base 222 of the housing 220, and mayextend upwards from the isolated base 222. The isolated base 222 may bea rigid, solid plate and may include various apertures to allowfastening devices to be inserted therethrough. Each of the blades 202may be attached to the isolated base 222 by brackets 224 and fasteners226. The isolated base 222 may have a substantially square geometry andthe blades 202 may be oriented along the isolated base 222 such that thewidth (e.g., the width 314 of FIG. 3 ) of each blade is aligned with adiagonal axis across the isolated base 222 (e.g., an axis extendingacross opposite corners of the isolated base 222). As shown in FIG. 2and FIG. 12 , the blades 202 do not contact one another.

The multi-detector system 200 is depicted in FIGS. 13 and 14 , from afront view and a perspective view, respectively, with the blades 202enclosed by the complete housing 220 (e.g., FIG. 2 shows only portionsof the housing 220). As such, the housing 220 also includes a top plate1302 coupled to top edges of two oppositely arranged side walls 1304.The top plate 1302 may be a rigid plate with a square geometry and mayhave similar dimensions to the isolated base 222. As shown in FIG. 13 ,a bottom face 1306 of the top plate 1302 may be in contact with the topedge 326 of the plate 206 of each blade 202. The objective 208 of eachblade 202 may protrude above a top surface 1308 of the top plate 1302,between the top surface 1308 and the plate holder 700, when the plateholder 700 is positioned over the objectives 208. As shown in FIG. 14 ,the stage 1150, configured to adjust the position of the plate holder700 along the top plate 1302, is coupled to the plate holder 700.

As depicted in FIG. 13 , the objectives 208 extend upwards from theblades 202 into a space between the top plate 1302 and the plate holder700 but does not contact the plate holder 700 or the microplate (e.g.,the microplate 600 of FIGS. 6-7 and 9-10 ) supported by the plateholder. The plate holder 700 may be translated along the x-y planewithout obstacles impeding movement of the plate holder 700. A distancethat the plate holder 700 is vertically spaced away from the objectives208 may be configured to allow the objectives 208 to be positioned at atarget distance from the microplate 600, located above, and a targetdistance from the tube lenses (e.g., the tube lens 404 of FIG. 4 ),located below. By placing the objectives 208 at the target distancesfrom the microplate 600 and the tube lenses, a maximum FOV andresolution may be obtained from the quantitative microscopy assembliesof the multi-detector system 200.

The plate holder 700 may be coupled to the stage 1150, the stage 1150also positioned above the top plate 1302 of the housing 220 and attachedto the top plate 1302. As shown in FIGS. 11A, 11B and 14 , the plateholder 700 may include a portion that extends onto and over the stage1150, forming a bracket 1410. As shown in FIG. 14 , the bracket 1410 maybe secured to the stage 1150 via fasteners, thereby fixedly coupling thestage 1150 to the plate holder 700. The stage 1150 is arranged alongsidethe plate holder 700 (with the exception of the bracket 1410 of theplate holder 700) such that the stage 1150 is located beside themicroplate 600 when the microplate 600 is positioned in the opening(e.g., the opening 714 of FIG. 8 ) of the plate holder 700.

The stage 1150 may be a two-axis stage configured with bearings, such asmechanical bearings, air bearings, etc., to allow the plate holder 700to translate along each of the x-axis and the y-axis relative to theobjectives 208. In some examples, movement of the stage, and thereforeof the plate holder 700, may be controlled by a motor. In otherexamples, a relative position of the stage 1150 may be manuallyadjusted. Adjustment of the positioning of the stage 1150 allows theFOVs of the objective 208 to be modified with respect to the microplate600, allowing complete imaging of the microplate 600.

The side walls 1304 of the housing 220 extend between the top plate 1302and the isolated base 222 on opposite sides of the top plate 1302 andthe isolated base 222. The side walls 1304 may be similarly configuredto one another and, as shown in FIG. 2 , may include openings 228 toallow the blades 202 to be easily accessed through the openings 228. Asillustrated in FIG. 14 , the openings 228 may be hidden by removablecovers 1402 to block undesired entry of external objects through theopenings 228. A height 1305 (indicated in FIG. 13 ) of the side walls1304 may be similar to the height 312 of the blades 202, as shown inFIG. 3 , to enable protrusion of the objectives 208 above the top plate1302 of the housing 220.

As shown in FIG. 13 , the blades 202 may be spaced away from the sidewalls 1304 so that the blades 202 do not contact the side walls 1304.Each of the blades 202 may be oriented similarly around the central axis204 such that the front side 210 of one the blades 202 faces the backside 212 of the blade in front and the back side of the blade faces thefront side 210 of the blade behind. Furthermore, the blades 202 areoriented with the inner side 302 of each blade proximate to the centralaxis 204. As such, the objectives 208 of the blades 202 are clusteredaround the central axis 204 at an upper region of the multi-detectorsystem 200 and spaced apart as described above with reference to FIG. 12.

The orientation of the blades 202 also positions the notched section 316of the inner side 302 of each of the blades proximate to the centralaxis 204. As illustrated in FIG. 14 , clustering of the notched section316 of each blade 202 around the central axis 204 forms an area of spacein which a central fan 1404 may be located. The multi-detector system200 is shown resting on a chassis 1406 in FIG. 14 , the chassis 1406including a bottom plate 1408. The chassis 1406 may support peripheralcomponents such as electronic devices coupled to the multi-detectorsystem 200.

As shown in FIG. 15 , the central fan 1404 may be coupled to the bottomplate 1408 of the chassis 1406 and extend upwards, along the z-axis fromthe bottom plate 1408, through an opening 1601 in the isolated base 222of the housing 220, the opening 1601 illustrated in FIG. 16 . Theopening 1601 in the isolated base 222 may be sized such that edges ofthe opening do not contact the central fan 1404. Vibrations from thecentral fan 1404 are therefore not propagated through the housing 220and blades 202 of the multi-detector system 200. As shown in FIG. 15 ,the central fan 1404 has a lower portion 1502, the lower portion 1502extending between the bottom plate 1408 and the isolated base 222, andan upper portion 1504, the upper portion 1504 protruding above theisolated base 222. The lower portion 1502 of the central fan 1404 may bea cylindrical duct with a plurality of slots 1506. The plurality ofslots 1506 may fluidically couple air outside of the lower portion 1502of the central fan 1404 to air inside of the lower portion 1502 of thecentral fan 1404.

The upper portion 1504 includes a mount 1508 and an impeller 1602, asshown in FIG. 16 in a top view of the central fan 1404. The mount 1508may have a square outer geometry and may include vertical walls, asshown in FIGS. 14 and 15 , that surround the impeller 1602 and fasteners1604 that secure the mount 1508 to the isolated base 222. A hub 1606 ofthe impeller 1602 may be rotatably coupled to the mount 1508. A motordriving rotation of the impeller 1602 may be arranged below the hub1606, under the isolated base 222 and enclosed by the lower portion 1502of the central fan 1404.

When the central fan 1404 is actuated, e.g., by operation of the motor,rotation of the impeller 1602 draws air laterally into the lower portion1502 of the central fan 1404 from a space between the isolated base 222and the bottom plate 1408 of the chassis 1406, as indicated in FIG. 15by arrows 1510. The air is pulled into the lower portion 1502 throughthe plurality of slots 1506 and pushed upwards, along the central axis204, driving air flow across surfaces of the blades 202, as indicated byarrows 1512.

As the air flow upwards, heat is extracted from components of the blades202 arranged along the inner side 302, such as the emission filter 402and the tube lens 404 depicted in FIG. 4 . The air may also cool thecamera mount 408. By arranging the central fan 1404 at a central regionof the isolating base 222 of the multi-detector system 200, enhancedthermal management of heat-generating and heat-sensitive components isenabled. The multi-detector assembly may further include a conventionalcooling system, such as a fan mounted along a rear wall of an outercasing of the multi-detector system 200 (not shown).

As shown in FIGS. 2, 13, and 14 , the housing 220 of the multi-detectorsystem 200 may also include vibration isolators 230 coupled to theisolated base 222 of the housing 220 and interfacing with the bottomplate 1408 of the chassis 1406. The vibration isolators 230 may besupports upon which the housing 220 of the multi-detector system 200sits. The vibration isolators 230 may have L-shaped profiles with aplanar base 232, or foot 232, that directly contacts the bottom plate1408 of the chassis 1406 of FIG. 14 . Vertical portions 234 (e.g.,aligned with the z-axis) of the vibration isolators 230 may extendthrough a corresponding opening in the isolated base 222 and may befitted with a fastener to secure the vibration isolators 230 to theisolated base 222.

The multi-detector system 200 is depicted with three vibration isolators230 herein but may include more of the vibration isolators 230 in otherexamples. As shown in FIGS. 2 and 14 , two of the vibration isolators230 are positioned proximate to a first edge 236 of the isolated base222 and aligned with one another along the first edge 236. The twovibration isolators 230 are spaced apart from one another such that eachof the vibrations isolators 230 is proximate to one of the side walls1304. The feet 232 of the two vibration isolators 230 may be orientedsuch that a length of the feet 232 (e.g., a longest dimension of thefeet 232) is parallel with the y-axis and extending from the verticalportions 234 in a direction away from the blades 202.

As shown in FIG. 13 , a third of the vibration isolators 230 (e.g., athird vibration isolator 231) may be positioned along a second edge 1310of the isolated base 222, opposite of the first edge 236. The thirdvibration isolator 231 may be located in a central region along thesecond edge 1310 of the isolated base 222, as illustrated in FIG. 13 .The foot 232 of the third vibration isolator 231 may be orientedperpendicular relative to the feet 232 of the two vibration isolators230 positioned along the first edge 236 of the isolated base 222, with alength of the foot 232 of the third vibration isolator 231 parallel withthe x-axis.

At least a portion of the vibration isolators 230 may be formed of aflexible, dampening material, such as rubber. Vibrational motiongenerated at the electronic components supported by the chassis 1406, asshown in FIG. 14 , and transmitted through the chassis 1406 maytherefore be absorbed by the vibration isolators 230. The housing 220and the blades 202 of the multi-detector system 200 are thereby isolatedfrom the vibrational motion, thus reducing noise in the imaging data.The positioning of the vibration isolators 230 along the isolated base222 and the orientation of the feet 232 provide stability to the housing220 such that the multi-detector system 200 remains level and resistantto tipping while minimizing points of contact between the bottom plate1408 of the chassis 1406 and the isolated base 222 through whichoscillating motions may be transmitted.

Imaging of the microplate by the multi-detector assembly may rely uponan ability of each quantitative microscopy assembly to rapidly focus ona suitable focal point along a depth of the microplate, e.g., withrespect to the z-axis. However, a presence of multiple interfacesbetween samples, located in the wells of the microplate, and theobjectives may confound a process for selecting a correct interface forobtaining high resolution images of the samples. To address this issue,a process for adjusting the focus of each objective of themulti-detector assembly may be implemented at a controller of themulti-detector assembly, such as the controller 124 of FIG. 1 . Theprocess is described below with reference to FIGS. 17-24 .

The process may include dividing the microplate, e.g., the microplate600 of FIGS. 6-7 and 9-10 , into four equally-sized quadrants, eachquadrant including 24 wells, such as the wells 610 of the microplate600. For each of the wells, multiple images may be obtained before acomplete image of each well is achieved. For example, a top down view ofone well 610 is shown in FIG. 17 . When preparing to execute imaging ofthe well 610, the process may include overlaying a grid 1702 with thewell 610 to provide an array of regions upon which the objective may bealigned. Images of each section of the grid 1702 may be captured andcompiled to provide a complete image of the well 610.

While a focus of a corresponding objective on each section of the grid1702 may be adjusted by the stage, e.g., the stage 1150 of FIGS. 11A,11B, 13 and 14 , the focus of the objective along the depth of the well610, e.g., along the z-axis, may be modified by varying a height of theobjective. For example, a distance along the z-axis between theobjective and the microplate may be modulated by adjusting the verticalposition of the objective, e.g., by an automated or manual mechanism asdescribed above. The vertical adjusting of the objective may beconducted to align the focus of the objective with a target interface ofthe microplate.

Interfaces present in the microplate 600 are depicted in a diagram shownin FIG. 18 illustrating a side view of the well 610 and the objective208 positioned below the well 610. As the well 610 (and the microplatein which the well 610 is disposed) is held stationary with respect tothe z-axis, a distance 1802 between a bottom surface 1804 and a tip 1806of the objective 208 may be varied by modifying the vertical position ofthe objective 208. Adjustment of the vertical position of the objective208 may accommodate variations in a target interface for obtainingimages across the microplate 600.

The well 610 includes a base 1808 forming a floor of the well 610, wherethe bottom surface 1804 of the well 610 is also the first interface 1804of the base 1808. The first interface 1804 may be an interface betweenthe base 1808 and air, e.g., air external to and surrounding theobjective 208. The base 1808 also includes a second interface 1810, thesecond interface 1810 positioned above the first interface 1804. Thesecond interface 1810 may be an interface between an upper surface 1812of the base 1808 and a sample 1814 placed in the well 610. The sample1814 may be, in one example, a biological specimen configured tofluoresce in response to illumination by incident light.

The base 1808 may be formed of a transparent material, such as plasticor glass, that does not interfere with light transmission. A targetfocal depth of the objective 208 may be located at the second interface1810 to obtain images of the sample 1814 with minimal distortion andinterference from reflected light. Focusing of the objective 208 at thesecond interface 1810 may rely on the LAF, e.g., the LAF sensor 342 ofFIGS. 3-4 , of each quantitative microscopy assembly of themulti-detector assembly and algorithms, hereafter, autofocus algorithms,for adjusting the vertical position of the objective 208, the algorithmsstored at a memory of the controller and executed by the controller whenimages of the microplate are to be collected.

In contrast to conventional alignment of the objective 208, wherealignment of the objective 208 refers to adjustment of the verticalposition of the objective 208 to align the focus of the objective 208 atthe target interface, the objective 208 may be aligned mechanically. Bymechanically aligning the objective, a more robust method for separatingLAF reflection signals from sample signals is enabled. For example, thelaser beam generated by the LAF sensor may be at least partiallyreflected at the first interface 1804 and focusing of the objective 208on the second interface 1810 may only occur upon identification of whichsignals received by the detector, e.g., the camera 338 of FIGS. 3-4 ,are undesirable signals generated by reflection at the first interface1804 and which signals are target signals reflected by the secondinterface 1810.

A faster and more reliable process for focusing the objective on thesecond interface 1810 may be provided in combination with the autofocusalgorithms. The autofocus algorithms allows the target focal plane to befound and to be utilized to align the focus of the objective 208, basedon a laser light shape which may be viewed at an entry aperture of theobjective 208. In one example, autofocusing of the objectives may relyon triangulation with oblique illumination. For example, a first,conventional light shape 1900 is shown in FIG. 19 for comparison with asecond light shape 2000 shown in FIG. 20 . The second light shape 2000may be used in the multi-detector system 200 to align the focus of eachof the objectives. The light shapes may be produced by passing the laserbeam of the LAF sensor to an aperture stop or one or more half-moonmasks, which may bisect the beam. In one example, the bisected beam mayform an image of a half-circle when reflected from the microplate.

Turning first to FIG. 19 , the first light shape 1900 includes anobjective entry aperture 1902 overlaid with a first LAF beam 1904, theLAF beam 1904 represented as a shaded half-circle. A sharp edge 1906 ofthe first LAF beam 1904 is aligned with a center of the objective entryaperture 1902. The first LAF beam 1904 therefore overlaps with half ofan area of the objective entry aperture 1902 when the objective focus isaligned with a target focal plane. As an example, the user may predefinean offset between a reference plane at the target focal plane. A pair ofoffset adjustment lenses may be used to maintain the objective focus atthe offset from the target focal plane. When the objective focus is outof focus, e.g., not aligned with the target focal plane at themicroplate, the first light shape 1900 may be altered. For example, ashape, size, or alignment of the first light shape 1900 may change,thereby demanding vertical adjustment of the objective position.

In some examples, a refractive index of the base of the microplate wellmay not enable differentiation between reflections from an interface ofthe base corresponding to the target focal plane (e.g., the secondinterface 1810 of FIG. 18 ) from another interface of the base, (e.g.,the first interface 1804 of FIG. 18 ). To address this issue, a modifiedlight shape may be used instead for autofocusing the objectives of themulti-detector system 200.

For example, turning to the second light shape 2000 of FIG. 20 , theobjective entry aperture 1902 may also be overlaid with a second LAFbeam 2002. The second LAF beam 2002 has a smaller area than the firstLAF beam 1904 and a sharp edge 2004 of the second LAF beam 2002 is notaligned with the center of the objective entry aperture 1902. Instead,the second LAF beam 2002 overlaps with less than half of the objectiveentry aperture 1902. In one example, the second LAF beam 2002 mayoverlap with one fifth of the area of the objective entry aperture 1902.As described above, the second light shape 2000 of FIG. 20 may provideincreased separation between light reflected from the first interface1804 and light from the second interface 1810 of FIG. 18 .

For example, a smaller light shape area may increase a separationbetween reflected laser light sports from each interface of themicroplate well base. A first reflection of the laser beam representsreflection from the first interface 1804 and a second reflectionrepresents reflection from the second interface 1810. The increasedseparation between the first and second reflections arising from use ofthe smaller light shape results in a greater range through which theobjective may be out of focus and still successfully be autofocused on atarget interface, e.g., the second interface 1810.

The enhanced ability to identify reflected light resulting from themechanical alignment depicted in FIG. 20 enables high speed focusing ofthe objective on the second interface 1810 of FIG. 18 . The autofocusalgorithms may include setting two autofocus ranges: a first rangeconfigured to reach an air/base interface, such as the first interface1804 of FIG. 18 , and a second range configured to reach a base/sampleinterface, such as the second interface 1810 of FIG. 18 , where the baserefers to a base of a microplate well, such as the base 1808 of FIG. 18. The second range may be set at a farther distance from the objectivethan the first range, e.g., the second range is above the first range,and may be selected as a target range which includes the target focalplane of the objective at the base/sample interface. Image collectionmay be initiated only when the objective focus is adjusted to targetfocal plane within the second range which may be determined based on thelaser beam emitted by a laser source of the LAF sensor of each blade ofthe multi-detector assembly, such as the LAF sensor 342 of FIGS. 3-4 .The laser beam is reflected from the microplate and directed to adetector (such as a CCD camera) of the LAF sensor 342 to produce a lightshape image, such as the second light shape 2000 of FIG. 20 . The lightshape image generated by the LAF sensor may be used to triangulate aposition of the objective relative to the base of the microplate well.

For example, variations in a sensor readout of the LAF sensorcorresponding to relative objective position is plotted in a first graph2100 and a second graph 2200 in FIGS. 21 and 22 , respectively. Thesensor readout may be a unit-less value that is correlated with adistance of the objective, e.g., along the z-axis in FIGS. 3-4 , from aposition where the objective is in-focus. In one example, a minimumvalue for the sensor readout may be −512 and a maximum value may be+512. The sensor readout value increasing upwards along the y-axis ofthe first and second graphs 2100, 2200. When the sensor readout valueequals zero, the objective is either in-focus or so far out of focusthat the sensor is not detecting the reflected laser beam.

Along the abscissa in the first and second graphs 2100, 2200, therelative objective position moves upwards, towards the sample, to theright. The relative objective position may be a position of theobjective along an optical axis of the objective, e.g., along the z-axisin FIGS. 3-4 , below a sample or sample holder, e.g., a floor of amicroplate well. The first graph 2100 may be representative of datacollection and processing at the detector of the LAF sensor for theobjective focus alignment based on the first light shape 1900 of FIG. 19(e.g., conventional alignment) and the second graph 2200 may berepresentative of data collection and processing of the objective focusalignment based on the second light shape 2000 of FIG. 20 .

As shown in the first graph 2100 of FIG. 21 , the first range (e.g., ofthe air/base interface) is indicated along the abscissa by arrow 2102and the second range (e.g., of the base/sample interface) is indicatedby arrow 2104. A first plot 2106 of the first graph 2100 depicts achange in sensor readout value according to the relative objectiveposition through each of the first range 2102 and the second range 2104.For example, at a first point, P1, the objective is at a greatestallowable distance away from the sample and the sensor readout value isat the minimum.

Between a point P1 along the abscissa and a second point P2, theobjective may be below the air/base interface and the sensor readoutvalues are negative. The objective focus is aligned with the air/baseinterface when the sensor readout value is equal to zero at the secondpoint P2. Between the second point P2 and a third point P3 along theabscissa, the objective focus moves above the air/base interface butremains below the base/sample interface, causing the sensor readoutvalue to increase to the maximum value at the third point P3 and plateauat S2 between the third point P3 and a fourth point P4. At the plateau,the sensor may detect that the objective is approaching a surface butcannot determine how close the objective is to the surface.

At a fourth point P4 along the abscissa, the objective focus becomesaligned with the base/sample interface and the sensor readout valuedrops abruptly to zero. Between the fourth point P4 and a fifth pointP5, the objective focus moves above the base/sample interface and thesensor readout value remains at the maximum until the objective positionreaches the fifth point P5, which may be an end point of the secondrange. The sensor power drops to zero power at the fifth point P5 due tothe objective moving out of focus. A distance between P2 and P4, withrespect to the relative objective position, is indicated by arrow 2108.

As described above, the sensor readout value may be at zero either whenthe objective focus is aligned with one of the interfaces or when theobjective is out of focus, e.g., outside of each of the first range andthe second range. The sensor may be configured to distinguish betweenalignment with an interface and being out of focus by monitoring achange in the sensor readout value when the objective is shifted up ordown relative to position when the sensor readout value is zero. Forexample, if the sensor readout value remains at zero when the objectiveis moved up or down, the sensor may determine that the objective is outof focus. Alternatively, if the sensor readout value changes in responseto moving the objective up or down, the sensor may determine that theobjective is in or near a position where the objective is in-focus.

In the second graph 2200 of FIG. 22 , the first range is indicated alongthe abscissa by arrow 2202 and the second range is indicated by arrow2204. As described above, the objective is within each of the first andthe second ranges when sensor readout value is between the minimum valueand the maximum value and in-focus within each range when a second plot2206 of the second graph 2200 crosses zero. At a first point P1 alongthe abscissa, the objective focus is below the air/base interface andthe sensor readout value is at the minimum. The sensor readout valueincreases between the first point P1 and a second point P2, at which theobjective focus is aligned with the air/base interface and the sensorreadout value is at zero. Between the second point P2 and a third pointP3, the objective focus is above the air/base interface and the sensorreadout value increases to the maximum value and plateaus at the maximumvalue between the third point P3 and a fourth point P4. At the plateau,the sensor may detect that the objective is approaching a surface butcannot determine how close the objective is to the surface. At thefourth point P4, the objective focus reaches a beginning of the secondrange, below the base/sample interface, and the sensor readout valuedrops to the minimum value.

When the objective focus is within the second range, as indicated byarrow 2204, the sensor power increases between the fourth point P4 and afifth point P5 at which the objective focus is aligned with thebase/sample interface and the sensor readout value is zero. The sensorpower rises to the maximum as the objective focus moves above thebase/sample interface between the fifth point P5 and a sixth point P6.Between the sixth point P6 and a seventh point P7, the seventh point P7defining an end point of the second range, the sensor power plateausagain at the maximum value until dropping to zero power at the seventhpoint P7. Beyond the seventh point P7, the objective is out of focus. Adistance between P2 and P5, with respect to the relative objectiveposition, is indicated by arrow 2208.

Comparison of the first graph 2100 to the second graph 2200 shows thatthe distance between the alignment of the objective focus with theair/base interface and with the base/sample interface is greater in thesecond graph 2200 than in the first graph 2100. Thus the objective maymove a greater distance in between focusing on the air/base interfaceand the base/well interface, enabling the interfaces to bedifferentiated more robustly.

A method for adjusting the focus of the objective to align with thebase/sample interface (e.g., the second interface 1810 of FIG. 18 ) isdepicted in a view of relative objective position in FIG. 23 and a blockdiagram in FIG. 24 and will be described in conjunction with oneanother. The method may be stored in a memory of the controller (e.g.,the controller 124 of FIG. 1 ), included in the autofocus algorithms, aswell as instructions for carrying out the method by the controller. Thecontroller may execute the method based on the LAF sensor of each bladeof the multi-detector assembly and/or based on commands input by anoperator.

As shown in FIG. 23 , the objective 208 is positioned at varyingdistances below the well 610 of the microplate 600 according to eachstep of the method. The method may begin with the objective at a nominalposition, as shown at a first step 2302 of FIG. 23 . The nominalposition may be, for example, a resting position that the objectivereturns to when the multi-detector system is turned off, when themicroplate 600 is removed or replaced, and/or when image acquisition iscomplete. At the resting position, the objective may be positioned at alowest possible point relative to the height of the blade and a furtherdistance away from the microplate. The first step 2302 corresponds to2402 of FIG. 24 , at which the method includes confirming and/oradjusting the objective to the nominal position while positioned belowthe well 610 of the microplate 600.

At step 2304 of FIG. 23 and 2404 of FIG. 24 , the method includesraising the vertical position of the objective 208 until the focus ofthe objective is within the second range of indicated by arrow 2204 inFIG. 22 of the second interface 1810, the second interface 1810indicated in FIG. 23 . For example, the objective may be raised to therelative objective position corresponding to a left-most point of thesecond range indicated by arrow 2204 of FIG. 22 . Upon determining thatthe objective 208 is in the second range, the vertical position of theobjective 208 is further raised at 2406 of FIG. 24 , and shown at step2306 in FIG. 23 , until the objective 208 is beyond the second range,e.g., past the right-most point of the second range indicated by arrow2204 of FIG. 22 . The distance between the objective 208 and themicroplate 600 may be minimized.

The objective 208 is lowered at 2408 of FIG. 24 , also shown at step2308 of FIG. 23 , until the sensor power is within a target low powerand a target high power. For example, as shown in FIG. 22 , theobjective 208 may be lowered to a point within the second range (asindicated by arrow 2204) where the sensor power is between S1 and S2. Atstep 2310 of FIG. 23 , the objective 208 is lowered until the sensorpower is at zero. At 2410 of FIG. 24 , the method includes using the LAFsensor to make fine adjustments to the vertical position of theobjective 208 until the second interface 1810 is in-focus. As shown atstep 2312 in FIG. 23 , the objective 208, in one example, may be loweredfurther to maximize a resolution and FOV of the objective 208. Images ofthe well 610 may be captured until sufficient data is collected. Forexample, the well 610 may be overlaid with the grid 1702 of FIG. 17 ,thereby demanding collection of 16 images to generate a complete imageof the well 610 while adjusting a horizontal position of the microplate600 (e.g., along the x-y plane) via the stage 1150 of FIGS. 11A, 11B,and 14 .

At 2412 of the method, as shown in FIG. 24 , the LAF sensor is disabled,e.g., deactivated, and the horizontal position of the microplate 600 isadjusted to align the objective 208 with a different well of themicroplate 600. With each new well, 2406 to 2412 of FIG. 24 and steps2306 to 2312 of FIG. 23 may be repeated. As such, a time for re-focusingthe objective when transitioned to a new well of the microplate forimage collection may be reduced while an accuracy of the LAF system,with respect to selecting the correct interface, is increased.Furthermore, each objective of the multi-detector assembly may captureimages concurrently. The method described above with reference to FIGS.23 and 24 , based on the second light shape 2000 of FIG. 20 , may beimplemented at each quantitative microscopy assembly at the same time,allowing parallel screening of each quadrant of the microplate.

In this way, a focus of each objective of the multi-detector system maybe rapidly aligned with the base/sample interface of a microplate well.The fast autofocusing of the objectives allows image collection andprocessing to be expedited, thereby enhancing an efficiency of themulti-detector system. By utilizing a laser light shape with a reducedarea, a separation between a focus range of the air/base interface andthe base/sample interface may be increased, allowing more efficientfocusing of the objective with the target interface (e.g., thebase/sample interface) using the autofocus system.

Autofocusing of the multi-detector system, e.g., the multi-detectorsystem 200 of FIGS. 2 and 12-14 , may occur independently at each bladeof the multi-detector system. However, generation of complete, cohesiveimages of a microplate may depend on synchronization of image datacollected concurrently at each quantitative microscopy assembly of themulti-detector system. A method 2500 for synchronizing imaging data fromeach detector of the multi-detector system is shown in FIGS. 25A-25B.Instructions for carrying method 2500 may be executed by a controller,such as the controller 124 of FIG. 1 , based on instruction stored on amemory of the controller and in conjunction with signals received fromsensors of the multi-detector system, such as the sensors describedabove. The controller may employ actuators of the multi-detector systemto adjust operation of the system, according to the methods describedbelow.

Turning first to FIG. 25A, at 2502, method 2500 includes receiving anindication that the microplate is adjusted to a position suitable forimaging. The indication may include, for example, confirmation that themicroplate is inserted into a plate holder of the multi-detector system,such as the plate holder 700 of FIGS. 7-11B, and 13-14 . The plateholder may include, as an example, a position sensor monitoring aposition of a ram, such as the ram 1104 of FIGS. 11A-11B, configured tomoderate a position of a swing arm of the plate holder. Adjustment ofthe ram to a retracted position may indicate that the microplate is inplace.

As another example, the indication that the microplate is ready forimaging may include receiving confirmation via a LAF sensor at eachblade of the multi-detector system, such as the LAF sensor 342 of FIGS.3-4 . For example, the LAF sensor may emit a laser beam to be reflectedfrom a surface of the microplate, such as a surface at a base of amicroplate well supporting a sample. If any of the sensors does notreceive a suitable power profile, it is determined that the microplatewell is not present or the sample is not present at the microplate welland the microplate may be adjusted to align the objective with the nextwell. If the suitable power profile is detected by the LAF sensor, apresence of the microplate may be confirmed and image collection mayproceed. In some examples, if a stage of the plate holder is shiftedthrough expected dimensions of the microplate and no suitable powerprofile is detected, an absence of the microplate may be confirmed andthe system may be place in a stand-by or shut-down mode.

At 2504, method 2500 includes receiving instructions from a userregarding selection of imaging protocols. For example, an exposure timeof each detector of the multi-detector assembly may be input at agraphical user interface (GUI) of the multi-detector system controller.A desired set of channels, where each channel is a target frequency orwavelength of light, for illuminating the microplate may be indicated bythe operator at the GUI.

Furthermore, the imaging protocol selection may include indicating howmany of the quantitative microscope assemblies of each blade of themulti-detector system are activated to capture imaging data. In someexamples, all four blades may be used for image collection. In otherexamples, less than four of the blades may be used to capture imagesfrom select quadrants of the microplate. For example, activation ofwhich quantitative microscope assemblies of the blades may be selectedbased on a distribution of samples across the microplate.

In some examples, the multi-detector system may be configured oroptimized to collect different types of quantitative images. Forexample, one or more of the detectors may be assigned to fluorescenceimaging while one or more of the detectors may be assigned to adifferent type of assay, or a different combination of fluorescentcolors. The detectors may include different filters, light sources,optical benefits, etc. The microplate may therefore be processed bydifferent techniques simultaneously, thereby increasing a flexibility ofthe multi-detector system for data collection. During instances wherespeed is prioritized, the detectors may be configured identically.

At 2508, method 2500 includes determining parameters of the microplate.For example, the operator may enter a type of microplate at the GUI,e.g., a 96-well microplate, a 12-well microplate, a 384-well microplate,etc. The controller may refer to a look-up table stored in memoryproviding physical parameters of each type of plate. As an example, thelook-up table may include a well volume, well diameter, a spacingbetween wells, plate dimensions, etc., specific to the type ofmicroplate. The parameters stored in the look-up table may determine adistance that the microplate is translated, e.g., along the x-y plane,to transition the objective focus between wells to be imaged. Thedistance may vary according to the microplate type. For example, themicroplate may be translated a greater distance from a central focalpoint of a first well to a central focal point of a second, adjacentwell of a 12-well microplate than a 96-well microplate. However, byreferring to the look-up table based on identification of themicroplate, a speed of transition between wells during imaging may besimilar regardless of microplate type.

The method includes, at 2510, auto-focusing each objective of theselected blades at a first well of the corresponding quadrant of themicroplate. Auto-focusing of the objectives is enabled via respectiveLAF sensors included in each blade of the multi-detector system. Forexample, the method depicted in FIG. 24 for adjusting the objectivefocus to align with a base/sample interface of the first well may beexecuted by a LAF controller, the LAF controller communicatively linkedto the system controller.

Autofocusing at each objective may also include, at 2512, optimizing aspeed at which positions of the objectives, e.g., along the z-axis, areadjusted according to a target positioned determined by the LAFcontroller. For example, variations in thickness amongst floors of eachwell of the microplate may result in variations in the target positionfor each objective. The speed of objective repositioning, e.g., asfacilitated by motors in objective modules such as the objective module328 of FIG. 3 , may be selected for each objective based on a desiredamount of time for the objectives to be adjusted to the targetpositions. As an example, a motor of a first objective of the objectivesthat is furthest displaced from it respective target position may becommanded to operate at a fastest allowable speed to place the objectivein the target position rapidly. The speed of the motors of the remainingobjectives may be selected based on the motor speed of the firstobjective such that the remaining objectives are positioned in theirrespective target positions no later than the first objective.

At 2514, method 2500 includes capturing image data at the first well ofeach quadrant corresponding to the objectives of the selected blades.Capturing image data may also include, at 2516, coordinating cyclingthrough the specified, e.g., selected, channels at the specifiedexposures for each collected image, e.g., based on the selectionreceived at 2504. For example, an image at each target focal point maybe obtained for each specified exposure. A period of time for capturinga complete suite of images may therefore vary depending on a number ofchannels and a number of different exposures selected. Further detailsof coordinating cycling through the specific channels are depicted atFIG. 26 .

Turning now to FIG. 26 , a method 2600 for capturing image data at eachobjective of the multi-detector system is shown. At 2602, method 2600includes confirming if activation of the light source and the camera areindicated. Activation of the light source and camera may be triggeredwhen the objective of each selected blade is adjusted to the targetposition to align the objective focus with the target focal plane, asdescribed above with reference to 2510 of method 2500. When eachobjective is at its respective target position, as determined based on aposition sensor of the objective module at each blade, the controllermay command activation of the light source and the camera of each blade.

If activation of the light source and the camera is not indicated,method 2600 continues to 2604 to maintain the light source and thecamera off, e.g., deactivated. As an example, one or more of theobjectives may not be at the target position and additional adjustmentto focus the objectives may be demanded. The method returns to 2602 toconfirm whether activation of the light source and the camera isindicated.

If activation of the light source and the camera is confirmed at 2602,method 2600 includes activating a first channel at the light source ofeach selected blade at 2606. In other words, a same LED at each lightsource is activated to emit a same frequency of light to illuminate themicroplate in unison. For example, the first LED 412 a of FIG. 4 may beactivated at each light source of the selected blades. By emitting lightfrom the same first channel at each of the blades synchronously, alikelihood of signal crosstalk is reduced.

At 2608, method 2600 includes capturing an image at each camera of theselected blades and storing the captured images. For example, thecaptured images may be stored at the controller's memory, e.g., at ahard drive, to retrieved for further processing and display. Method 2600includes confirming if another channel of the light sources is to beactivated for collecting further image data, according to the receivedselection of channels at 2504 of method 2500. If another channel is tobe activated, method 2600 returns 2606 to activate another channel atthe light sources of the selected blades. If no additional channels areto be activated, method 2600 includes deactivating the light sources andthe cameras at 2610 and method 2600 returns to method 2500 of FIG. 25A,e.g., at 2514 of method 2500. In instances where more than one exposureis selected for each channel, method 2600 may repeat for each selectedexposure before returning to method 2500.

Returning to method 2500, capturing image data at the first well of eachquadrant may also include collecting more than one image at the firstwell, and each subsequent well, of the microplate at 2518. For example,to obtain a complete image of the first well, the focus of eachobjective may be adjusted, e.g., via translation of the microplate by astage, to various focal points along the base of the well. In oneexample, an area of the first well may divided into a 16 by 16 grid, asshown in FIG. 17 , and the objective may be focused at each section ofthe grid. By capturing an image of each section of the grid, the gridimages may be compiled into a single, cohesive image of the first welland each successively imaged well. Method 2500 continues at method 2550of FIG. 25B.

Turning now to FIG. 25B, at 2520, method 2550 includes moving themicroplate such that the objectives are focused on a next well of therespective quadrant of the microplate. The new well may be a second welladjacent to the first well or a next closest well supporting a sample.At 2522, method 2550 includes adjusting the objective focus of eachselected blade at the next well using the respective LAF sensor, asdescribed with reference to FIG. 24 . Image data is collected at 2524for the next well of each quadrant corresponding to the selected blades.

At 2526, method 2550 includes confirming if additional wells of themicroplate are to be imaged. If images of one or more wells of themicroplate supporting a sample have not been collected or if imagecapture at each well of each quadrant is not complete, the methodreturns to 2520 to continue image data collection at a next well of eachquadrant. If no additional wells are to be imaged, e.g., all targetwells of each quadrant have been imaged, the method proceeds to 2528 tosave and/or display the image data. The image data may include fourcomplete images of four of the microplate wells, each image obtained byone of the objectives. For example, the images may be saved to adatabase stored in the controller's memory and additionally oralternatively saved to a server in a suitable file format. As anexample, the images may be saved in a format that includes metadata.Additionally or alternatively, the images may be displayed at the GUI.Method 2550 returns to the start of method 2500.

In this way, screening of samples on a microplate may be achievedrapidly and efficiently by a multi-detector quantitative microscopysystem. The multi-detector quantitative microscopy system (e.g., system)may include two or more microscope assemblies arranged in an x-shapedconfiguration along a base plate of the system. Each assembly may beconfigured as a blade including a plate supporting a set of imagingcomponents. Objectives may be located at tops of each blade, protrudingupwards through an opening in a housing of the system. The objectivesmay be clustered around a central axis of the system and spaced furtherapart along one horizontal axis than along a second, perpendicularhorizontal axis. Furthermore, the spacing between the objectives may beselected to enable all objectives to collect images simultaneously,within a targeting imaging region of the microplate during each imagecollection cycle. As a result, a cycling frequency of microplate imagecollection may be faster than conventional systems, allowing livebiology events to be captured. Various components of the microscopeassemblies, such as laser autofocus sensors, a positioning of tubelenses with respect to the objectives with respect to a tube lens, anarrangement of light sources, etc., as well as of the overall system,such as a plate holder for supporting the microplate, may be optimizedto contribute to the rapid cycling frequency. Furthermore, a packagingof the system allows the system to have a compact footprint, therebyenabling a portability of the system and reducing its demand for space.

The following claims particularly point out certain combinations andsub-combinations regarded as novel and non-obvious. These claims mayrefer to “an” element or “a first” element or the equivalent thereof.Such claims should be understood to include incorporation of one or moresuch elements, neither requiring nor excluding two or more suchelements. Other combinations and sub-combinations of the disclosedfeatures, functions, elements, and/or properties may be claimed throughamendment of the present claims or through presentation of new claims inthis or a related application. Such claims, whether broader, narrower,equal, or different in scope to the original claims, also are regardedas included within the subject matter of the present disclosure.

1. An imaging system, comprising: a plurality of microscope assembliesarranged radially around a central axis of the imaging system, coupledto vertically oriented plates with a set of objectives arranged at topsof the plurality of microscope assemblies and wherein the plates areoriented to extend away from the central axis in a radial direction toform an x-shaped configuration.
 2. The imaging system of claim 1,wherein an inner side of each the plates is proximate to the centralaxis and each objective of the set of objectives is aligned with theinner side of one of the plates.
 3. The imaging system of claim 2,wherein the plurality of microscope assemblies includes tube lenses,each tube lens of the tube lenses positioned below one objective of theset of objectives along the inner side of one of the plates, insulatingcamera mounts located below the tube lenses, cameras coupled to thecamera mounts, laser auto-focus systems oriented parallel with a heightof the plates and aligned with an outer side of each of the plates, theouter side opposite of the inner side, and light sources coupled to acentral region of each of the plates, at front faces of the platesbetween the tube lenses and the laser auto-focus systems.
 4. The imagingsystem of claim 3, wherein the plurality of microscope assembliesfurther includes printed circuit board assemblies coupled to rear facesof the plates, the rear faces opposite of the front faces.
 5. Theimaging system of claim 1, wherein the plates are coupled to a baseplate of the imaging system and extend upwards from the base plate andwherein the coupling of the plates to the base plate is configured toincrease a spacing between the set of objectives along a firsthorizontal axis of the imaging system relative to a spacing between theset of objectives along a second horizontal axis, the second horizontalaxis perpendicular to the first horizontal axis.
 6. The imaging systemof claim 5, wherein the set of objectives are positioned in a rectanglealong a horizontal plane of the imaging system.
 7. The imaging system ofclaim 1, wherein the plurality of microscope assemblies includes atleast two microscope assemblies.
 8. The imaging system of claim 1,wherein the plurality of microscope assemblies includes four or moremicroscope assemblies.
 9. A multi-detector quantitative microscopysystem, comprising: two or more microscope assemblies orienteddiagonally along a square base plate of the multi-detector quantitativemicroscopy system with an inner side of each of the two or moremicroscope assemblies positioned at a central region of the square baseplate and an outer side of each of the two or more microscope assembliesproximate to a corner of the square base plate; and a set of objectivesarranged at a top of the multi-detector quantitative microscopy system,each of the set of objectives coupled to one of the two or moremicroscope assemblies along the inner side, wherein a spacing betweenthe set of objectives is greater along a first horizontal axis thanalong a second horizontal axis, perpendicular to the first horizontalaxis.
 10. The multi-detector quantitative microscopy system of claim 9,further comprising a set of feet coupled to a bottom face of the squarebase plate and extending between the square base plate and a chassis ofthe multi-detector quantitative microscopy system.
 11. Themulti-detector quantitative microscopy system of claim 10, wherein thefeet include a vertical portion and a horizontal portion coupled to anend of the vertical portion distal to the square base plate and whereinthe feet are configured to stabilize the multi-detector quantitativemicroscopy system and absorb vibrations.
 12. The multi-detectorquantitative microscopy system of claim 10, further comprising a centralfan coupled to the chassis and extending through an opening in thesquare base plate, the opening aligned with a central axis of themulti-detector quantitative microscopy system.
 13. The multi-detectorquantitative microscopy system of claim 12, wherein the central fanincludes an upper portion that protrudes above the square base plate,surrounded by the two or more microscope assemblies, and wherein thecentral fan is configured to draw air from below the square base plate,up through a lower portion of the central fan and out through the upperportion to cool the two or more microscope assemblies.
 14. Themulti-detector quantitative microscopy system of claim 13, wherein thelower portion of the central fan includes a plurality of slots to enablethe drawing of air from below the square base plate into the lowerportion of the central fan.
 15. A multi-detector imaging system,comprising: a set of objectives arranged around a central axis of themulti-detector imaging system, spaced apart by a greater distance alonga first horizontal axis than a second horizontal axis, each of the setof objectives coupled to a top portion of a microscope assembly, andwherein the set of objectives is configured to capture a portion of animage of a microplate synchronously to generate a complete image of themicroplate.
 16. The multi-detector imaging system of claim 15, whereinthe microplate is coupled to a plate holder positioned above the set ofobjectives and wherein a position of the microplate relative to the setof objectives is adjusted by a stage attached to the plate holder. 17.The multi-detector imaging system of claim 16, wherein the plate holderincludes a swing arm for maintaining a position of the microplate withinthe plate holder and a stage for adjusting the position of microplatealong a horizontal plane of the multi-detector imaging system.
 18. Themulti-detector imaging system of claim 15, wherein an imaging frequencyof the multi-detector imaging system is configured to capture transientsignals of live cells.
 19. The multi-detector imaging system of claim15, wherein a footprint of the multi-detector imaging system is similarto a footprint of an imaging system with a single detector.
 20. Themulti-detector imaging system of claim 15, wherein the set of objectivesincludes four objectives arranged in a rectangular configuration andprotruding through a ceiling of a housing of the multi-detector imagingsystem.